bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2020‒09‒06
forty-eight papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing
  1. Structural mechanism for amino acid-dependent Rag GTPase nucleotide state switching by SLC38A9.
  2. mTORC1 activation contributes to autophagy inhibition via its recruitment to lysosomes and consequent lysosomal dysfunction in cadmium-exposed rat proximal tubular cells.
  3. Lysosome as a Central Hub for Rewiring PH Homeostasis in Tumors.
  4. Mechanistic Target of Rapamycin (mTOR) Signaling Activation Antagonizes Autophagy to Facilitate Zika Virus Replication.
  5. Surface plasmon resonance analysis of complex formation of therapeutic recombinant lysosomal enzymes with domain 9 of human cation-independent mannose 6-phosphate receptor.
  6. Has resveratrol a potential for mucopolysaccharidosis treatment?
  7. Thrombin-activated PAR1 membrane expression is regulated by Rab11a-RCP complex dissociation.
  8. GM2 Gangliosidoses: Clinical Features, Pathophysiological Aspects, and Current Therapies.
  9. Engineering of a dual-site molecular probe for logical bioimaging of lysosomal H2S and pH.
  10. Molecular dynamics study with mutation shows that N-terminal domain (NTD) structural re-orientation in NPC1 is required for proper alignment of cholesterol transport.
  11. A red-emission fluorescent probe for visual monitoring of lysosomal pH changes during mitophagy and cell apoptosis.
  12. Therapeutic benefit after intracranial gene therapy delivered during the symptomatic stage in a feline model of Sandhoff disease.
  13. Autophagy under glucose starvation enhances protein translation initiation in response to re-addition of glucose in C2C12 myotubes.
  14. Ubiquitin, Autophagy and Neurodegenerative Diseases.
  15. Decanoic acid inhibits mTORC1 activity independent of glucose and insulin signaling.
  16. The Alzheimer's disease-associated C99 fragment of APP regulates cellular cholesterol trafficking.
  17. The DNA methyltransferase DNMT3A contributes to autophagy long-term memory.
  18. Distinct alpha-Synuclein species induced by seeding are selectively cleared by the Lysosome or the Proteasome in neuronally differentiated SH-SY5Y cells.
  19. Branched-chain ketoacid overload inhibits insulin action in the muscle.
  20. PDPK1 regulates autophagosome biogenesis by binding to PIK3C3.
  21. Enzyme Replacement Therapy for Lysosomal Storage Diseases.
  22. Amino acid starvation inhibits autophagy in lipid droplet-deficient cells through mitochondrial dysfunction.
  23. Changes in expressions of genes involved in the regulation of cellular processes in mucopolysaccharidoses as assessed by fibroblast culture-based transcriptomic analyses.
  24. Upregulation of Akt/Raptor signaling is associated with rapamycin resistance of breast cancer cells.
  25. Detailed epitope mapping of neutralizing anti-drug antibodies against recombinant α-galactosidase A in patients with Fabry disease.
  26. [Gaucher disease].
  27. The Roles of Ubiquitin in Mediating Autophagy.
  28. C-terminal tail length guides insertion and assembly of membrane proteins.
  29. Substrate metabolism regulated by Sestrin2-mTORC1 alleviates pressure overload-induced cardiac hypertrophy in aged heart.
  30. Antioxidant Modulation of mTOR and Sirtuin Pathways in Age-Related Neurodegenerative Diseases.
  31. MITF and TFEB cross-regulation in melanoma cells.
  32. Reconstitution of autophagosome nucleation defines Atg9 vesicles as seeds for membrane formation.
  33. Bergmeister's papilla in a young patient with type 1 sialidosis: case report.
  34. mTOR signaling regulates the morphology and migration of outer radial glia in developing human cortex.
  35. Identification of a missense ARSA mutation in metachromatic leukodystrophy and its potential pathogenic mechanism.
  36. An Experimental Model of Neurodegenerative Disease Based on Porcine Hemagglutinating Encephalomyelitis Virus-Related Lysosomal Abnormalities.
  37. Epr1, a UPR-upregulated soluble autophagy receptor for reticulophagy.
  38. Reconsolidation of a post-ingestive nutrient memory requires mTOR in the central amygdala.
  39. Minocycline inhibits mTOR signaling activation and alleviates behavioral deficits in the Wistar rats with acute ischemia stroke.
  40. Atg8-Family Proteins-Structural Features and Molecular Interactions in Autophagy and Beyond.
  41. Genetic Inactivation of Two-Pore Channel 1 Impairs Spatial Learning and Memory.
  42. The neurofibromatosis type I gene promotes autophagy via mTORC1 signalling pathway to enhance new bone formation after fracture.
  43. Assessing Lysosomal Disorders in the NGS Era: Identification of Novel Rare Variants.
  44. An Excitable Ras/PI3K/ERK Signaling Network Controls Migration and Oncogenic Transformation in Epithelial Cells.
  45. In-cell architecture of the nuclear pore and snapshots of its turnover.
  46. mTOR plays a pivotal role in multiple processes of enamel organ development principally through the mTORC1 pathway and in part via regulating cytoskeleton dynamics.
  47. TSC1 intragenic deletion transmitted from a mosaic father to two siblings with cardiac rhabdomyomas: Identification of two aberrant transcripts.
  48. ASAH1-related disorders: Description of 15 novel pediatric patients and expansion of the clinical phenotype.
  1. Nat Struct Mol Biol. 2020 Aug 31.
    Structural mechanism for amino acid-dependent Rag GTPase nucleotide state switching by SLC38A9.
    Simon A Fromm, Rosalie E Lawrence, James H Hurley.
      The Rag GTPases (Rags) recruit mTORC1 to the lysosomal membrane in response to nutrients, where it is then activated in response to energy and growth factor availability. The lysosomal folliculin (FLCN) complex (LFC) consists of the inactive Rag dimer, the pentameric scaffold Ragulator, and the FLCN:FNIP2 (FLCN-interacting protein 2) GTPase activating protein (GAP) complex, and prevents Rag dimer activation during amino acid starvation. How the LFC is disassembled upon amino acid refeeding is an outstanding question. Here we show that the cytoplasmic tail of the human lysosomal solute carrier family 38 member 9 (SLC38A9) destabilizes the LFC and thereby triggers GAP activity of FLCN:FNIP2 toward RagC. We present the cryo-EM structures of Rags in complex with their lysosomal anchor complex Ragulator and the cytoplasmic tail of SLC38A9 in the pre- and post-GTP hydrolysis state of RagC, which explain how SLC38A9 destabilizes the LFC and so promotes Rag dimer activation.
    DOI:  https://doi.org/10.1038/s41594-020-0490-9
  2. J Inorg Biochem. 2020 Aug 21. pii: S0162-0134(20)30259-2. [Epub ahead of print]212 111231
    mTORC1 activation contributes to autophagy inhibition via its recruitment to lysosomes and consequent lysosomal dysfunction in cadmium-exposed rat proximal tubular cells.
    Cai-Yu Lian, Heng Yang, Zhen-Zhen Zhai, Zi-Fa Li, Dian-Gang Han, Lin Wang.
      Autophagy dysregulation is implicated in cadmium (Cd)-induced nephrotoxicity. The mammalian target of rapamycin complex 1 (mTORC1) is a negative regulator of autophagy, but its role in Cd-induced autophagy inhibition and possible regulatory mechanisms remains poorly understood. In the present study, Cd exposure activated mTORC1 in primary rat proximal tubular (rPT) cells, and two mTORC1 inhibitors (rapamycin and torin 1) were separately utilized to inhibit Cd-induced mTORC1 activation. Data showed that Cd-inhibited autophagic flux was markedly restored by two mTORC1 inhibitors, respectively, as evidenced by immunoblot analysis of autophagy marker proteins and tandem red fluorescent protein-green fluorescent protein-microtubule associated protein light chain 3 (RFP-GFP-LC3) fluorescence microscopy assay. Importantly, Cd exposure triggered the recruitment of mTORC1 onto lysosome membrane assessed by immunofluorescence co-localization analysis, which was obviously inhibited by rapamycin or torin 1. Moreover, Cd-induced lysosomal alkalization, suppressed vacuolar ATPases (V-ATPases) protein levels and impaired lysosomal degradation capacity were markedly reversed by rapamycin or torin 1. In summary, these findings demonstrate that Cd recruits mTORC1 to lysosome membrane to induce its activation, which results in lysosomal dysfunction and resultant autophagy inhibition in rPT cells.
    Keywords:  Autophagy; Cadmium; Kidney; Lysosome; mTORC1; rPT cells
    DOI:  https://doi.org/10.1016/j.jinorgbio.2020.111231
  3. Cancers (Basel). 2020 Aug 27. pii: E2437. [Epub ahead of print]12(9):
    Lysosome as a Central Hub for Rewiring PH Homeostasis in Tumors.
    Ran Chen, Marja Jäättelä, Bin Liu.
      Cancer cells generate large quantities of cytoplasmic protons as byproducts of aberrantly activated aerobic glycolysis and lactate fermentation. To avoid potentially detrimental acidification of the intracellular milieu, cancer cells activate multiple acid-removal pathways that promote cytosolic alkalization and extracellular acidification. Accumulating evidence suggests that in addition to the well-characterized ion pumps and exchangers in the plasma membrane, cancer cell lysosomes are also reprogrammed for this purpose. On the one hand, the increased expression and activity of the vacuolar-type H+-ATPase (V-ATPase) on the lysosomal limiting membrane combined with the larger volume of the lysosomal compartment increases the lysosomal proton storage capacity substantially. On the other hand, enhanced lysosome exocytosis enables the efficient release of lysosomal protons to the extracellular space. Together, these two steps dynamically drive proton flow from the cytosol to extracellular space. In this perspective, we provide mechanistic insight into how lysosomes contribute to the rewiring of pH homeostasis in cancer cells.
    Keywords:  V-ATPase; lysosomal exocytosis; lysosome; pH regulation
    DOI:  https://doi.org/10.3390/cancers12092437
  4. J Virol. 2020 Sep 02. pii: JVI.01575-20. [Epub ahead of print]
    Mechanistic Target of Rapamycin (mTOR) Signaling Activation Antagonizes Autophagy to Facilitate Zika Virus Replication.
    Bikash R Sahoo, Aryamav Pattnaik, Arun S Annamalai, Rodrigo Franco, Asit K Pattnaik.
      Zika virus (ZIKV), a mosquito-transmitted flavivirus, is linked to microcephaly and other neurological defects in neonates and Guillain-Barré syndrome in adults. The molecular mechanisms regulating ZIKV infection and pathogenic outcomes are incompletely understood. Signaling by the mechanistic (mammalian) target of rapamycin (mTOR) kinase is important for cell survival and proliferation, and viruses are known to hijack this pathway for their replication. Here, we show that in human neuronal precursors and glial cells in culture, ZIKV infection activates both mTOR complex 1 (mTORC1) and 2 (mTORC2). Inhibition of mTOR kinase by torin1 or rapamycin results in reduction in ZIKV protein expression and progeny production. Depletion of Raptor, the defining subunit of mTORC1, by small interfering RNA (siRNA), negatively affects ZIKV protein expression and viral replication. Although depletion of Rictor, the unique subunit of mTORC2 or the mTOR kinase itself also inhibits the viral processes, the extent of inhibition is less pronounced. Autophagy is transiently induced early by ZIKV infection and impairment of autophagosome elongation by the Class III PI3K inhibitor 3-methyladenine (3-MA) enhances viral protein accumulation and progeny production. mTOR phosphorylates and inactivates ULK1 (S757) at later stages of ZIKV infection, suggesting a link between autophagy inhibition and mTOR activation by ZIKV. Accordingly, inhibition of ULK1 (by MRT68921) or autophagy (by 3-MA) reversed the effects of mTOR-inhibition, leading to increased levels of ZIKV protein expression and progeny production. Our results demonstrate that ZIKV replication requires the activation of both mTORC1 and mTORC2, which negatively regulates autophagy to facilitate ZIKV replication.IMPORTANCE The re-emergence of Zika virus (ZIKV) and its association with neurological complications necessitates studies on the molecular mechanisms that regulate ZIKV pathogenesis. The mTOR signaling cascade is tightly regulated and central to normal neuronal development and survival. Disruption of mTOR signaling can result in neurological abnormalities. In the studies reported here, we demonstrate for the first time that ZIKV infection results in activation of both mTORC1 and mTORC2 to promote the virus replication. Although autophagy is activated early in infection to counter virus replication, it is subsequently suppressed by the mTOR. These results reveal critical roles of mTOR signaling and autophagy in ZIKV infection and point to a possible mechanism underlying ZIKV-induced pathogenesis. Elucidating the role of mTOR signaling in ZIKV infection will provide insights into our understanding of the mechanisms of ZIKV-induced neurological complications and potential targets for therapeutic approaches.
    DOI:  https://doi.org/10.1128/JVI.01575-20
  5. Mol Genet Metab Rep. 2020 Dec;25 100639
    Surface plasmon resonance analysis of complex formation of therapeutic recombinant lysosomal enzymes with domain 9 of human cation-independent mannose 6-phosphate receptor.
    Minori Kanzaki, Takahiro Tsukimura, Yasunori Chiba, Hitoshi Sakuraba, Tadayasu Togawa.
      The efficacy of enzyme replacement therapy (ERT) for lysosomal storage diseases (LSDs) possibly depends on the cellular uptake of recombinant lysosomal enzymes (LEs), and it is known that cation-independent mannose 6-phosphate receptor (CI-M6PR) on the cell membrane is predominantly involved in the endocytosis of many LEs. To examine the biomolecular interaction between therapeutic LEs and CI-M6PR, we biophysically analyzed the complex formation of four LEs available with domain 9 of human CI-M6PR, a binding site of the receptor, by means of surface plasmon resonance (SPR) biosensor assays. The results revealed that the affinity of the LEs for domain 9 of the receptor increased in the following order: laronidase, agalsidase beta, idursulfase, and alglucosidase alfa; and the high affinity of laronidase for domain 9 of CI-M6PR was due to fast complex formation rather than slow dissociation of the complex. The affinity of the enzymes for domain 9 of CI-M6PR almost coincided with their cellular uptake. The SPR biosensor assay is sensitive and provides important information for the development of effective therapeutic LEs for LSDs.
    Keywords:  CD-M6PR, cation-dependent mannose 6-phosphate receptor.; CI-M6PR, cation-independent mannose 6-phosphate receptor; Cation-independent mannose 6-phosphate receptor; Complex formation; ERT, enzyme replacement therapy; Enzyme uptake; LE, lysosomal enzyme; LSD, lysosomal storage disease; Lysosomal enzyme; Lysosomal storage disease; M6P, mannose 6-phosphate; PBS, phosphate-buffered saline;; SPR, surface plasmon resonance; Surface plasmon resonance
    DOI:  https://doi.org/10.1016/j.ymgmr.2020.100639
  6. Eur J Pharmacol. 2020 Aug 30. pii: S0014-2999(20)30626-9. [Epub ahead of print] 173534
    Has resveratrol a potential for mucopolysaccharidosis treatment?
    Estera Rintz, Karolina Pierzynowska, Magdalena Podlacha, Grzegorz Węgrzyn.
      Mucopolysaccharidoses (MPS) represent a devastating group of lysosomal storage diseases (LSD) affecting approximately 1 in 25,000 individuals, where degradation of glycosaminoglycans (GAG) by lysosomal enzymes is impaired due to mutations causing defects in one of GAG-degrading enzymes. The most commonly used therapy for MPS is enzyme replacement therapy, consisting of application of an active form of the missing enzyme. However, supply of the missing enzyme is not enough in case of MPS types whose symptoms are expressed in central nervous system (CNS), as enzyme does not cross the blood-brain barrier. Moreover, even though enzyme replacement therapy for non-neuronopathic MPS IVA type is approved, it has a limited impact on bone abnormalities, that are one of main symptoms in the disease. Therefore, research into alternative therapeutic approaches for these types of MPS is highly desirable. One such alternative strategy is accelerated degradation of GAG by induction of autophagy. Autophagy is a process of lysosomal degradation of macromolecules that become abnormal or unnecessary for cells. One of the latest discoveries is that GAGs can also be such molecules. Potential drug should also cross blood-brain barrier and be safe in long-term therapy. It seems that one of the polyphenols, resveratrol, can meet the requirements. The mechanism of its action in autophagy stimulation is pleiotropic. Therefore, in this review, we will briefly discuss potential of resveratrol treatment for mucopolysaccharidosis through autophagy stimulation based on research in diseases with similar outcome.
    Keywords:  Autophagy; Glycosoaminoglycans; Mucopolysaccharidosis; Trans-Resveratrol
    DOI:  https://doi.org/10.1016/j.ejphar.2020.173534
  7. Cell Signal. 2020 Aug 26. pii: S0898-6568(20)30225-4. [Epub ahead of print] 109748
    Thrombin-activated PAR1 membrane expression is regulated by Rab11a-RCP complex dissociation.
    Alvarez-Arce Alejandro, Lee-Rivera Irene, López Edith, López-Colomé Ana María.
      PAR1 activation by thrombin promotes intracellular signaling leading to RPE cell transformation, proliferation, and migration, characteristic of fibroproliferative eye diseases. Due to the cleavage of PAR1 N-terminal domain, carried by thrombin, the arrest of PAR1 signaling is achieved by transport into lysosomes and degradation. Recent findings suggest that the GTPase Rab11a in conjunction with its effector RCP may direct PAR1 to lysosomes. Hereby we demonstrate that thrombin-induced PAR1 internalization and lysosomal targeting requires the disassembly of the Rab11a/RCP complex, and that this process depends on thrombin-induced intracellular calcium increase and calpain activation. These findings unveil a novel mechanism that regulates thrombin activated PAR1 internalization and degradation.
    Keywords:  Calpain; GPCR internalization; Proliferative disease; Retina; Thrombin; Trafficking
    DOI:  https://doi.org/10.1016/j.cellsig.2020.109748
  8. Int J Mol Sci. 2020 Aug 27. pii: E6213. [Epub ahead of print]21(17):
    GM2 Gangliosidoses: Clinical Features, Pathophysiological Aspects, and Current Therapies.
    Andrés Felipe Leal, Eliana Benincore-Flórez, Daniela Solano-Galarza, Rafael Guillermo Garzón Jaramillo, Olga Yaneth Echeverri-Peña, Diego A Suarez, Carlos Javier Alméciga-Díaz, Angela Johana Espejo-Mojica.
      GM2 gangliosidoses are a group of pathologies characterized by GM2 ganglioside accumulation into the lysosome due to mutations on the genes encoding for the β-hexosaminidases subunits or the GM2 activator protein. Three GM2 gangliosidoses have been described: Tay-Sachs disease, Sandhoff disease, and the AB variant. Central nervous system dysfunction is the main characteristic of GM2 gangliosidoses patients that include neurodevelopment alterations, neuroinflammation, and neuronal apoptosis. Currently, there is not approved therapy for GM2 gangliosidoses, but different therapeutic strategies have been studied including hematopoietic stem cell transplantation, enzyme replacement therapy, substrate reduction therapy, pharmacological chaperones, and gene therapy. The blood-brain barrier represents a challenge for the development of therapeutic agents for these disorders. In this sense, alternative routes of administration (e.g., intrathecal or intracerebroventricular) have been evaluated, as well as the design of fusion peptides that allow the protein transport from the brain capillaries to the central nervous system. In this review, we outline the current knowledge about clinical and physiopathological findings of GM2 gangliosidoses, as well as the ongoing proposals to overcome some limitations of the traditional alternatives by using novel strategies such as molecular Trojan horses or advanced tools of genome editing.
    Keywords:  GM2 gangliosidoses; Sandhoff disease; Tay–Sachs disease; lysosomal storage disorders; therapeutic alternatives; β-Hexosaminidases
    DOI:  https://doi.org/10.3390/ijms21176213
  9. Talanta. 2020 Nov 01. pii: S0039-9140(20)30577-4. [Epub ahead of print]219 121286
    Engineering of a dual-site molecular probe for logical bioimaging of lysosomal H2S and pH.
    Yongchao Liu, Yingjun Zhang, Xiao-Bing Zhang.
      Lysosomes contains numerous enzymes and proteins closely linked with cellular metabolism. The variation of lysosomal pH is closely related to cell homeostasis while hydrogen sulfide (H2S) has been regarded as an important gasotransmitter. Herein, via rational design, a high-sensitivity fluorescent probe ANp-Rh-Lys was synthesized for logical detection and bioimaging of H2S and pH in lysosomes of living cells. The probe showed different fluorescence signals towards H2S and lysosomal pH. Significantly, ANp-Rh-Lys is membrane-permeable and suitable for visualization of both pH variations and endogenous H2S in lysosomes. This logical design strategy may have great potential for detection of multiple analytes in complicated biological systems.
    Keywords:  Dual-site; Engineering; Fluorescent probe; Logical bioimaging
    DOI:  https://doi.org/10.1016/j.talanta.2020.121286
  10. J Neurochem. 2020 Aug 16.
    Molecular dynamics study with mutation shows that N-terminal domain (NTD) structural re-orientation in NPC1 is required for proper alignment of cholesterol transport.
    HyeJin Yoon, Hyunah Jeong, Hyung Ho Lee, Soonmin Jang.
      The lysosomal membrane protein NPC1 (Niemann-Pick type C1) and NPC2 (Niemann-Pick type C2) are main players of cholesterol control in the lysosome and it is known that the mutation on these proteins leads to the cholesterol trafficking related neurodegenerative disease, which is called the Niemann-Pick disease type C (NPC) disease. The mutation R518W or R518Q on the NPC1 is one of type of disease-related mutation that causes cholesterol transports to be cut in half, which results in the accumulation of cholesterol and lipids in the late endosomal/lysosomal compartment of the cell. Even though there has been significant progress with understanding the cholesterol transport by NPC1 in combination with NPC2, especially after the structural determination of the full length NPC1 in 2016, many details such as the interaction of the full length NPC1 with the NPC2, the molecular motions responsible for the cholesterol transport during and after this interaction, and the structure and the function relations of many mutations are still not well understood. In this paper, we report the extensive molecular dynamics simulations in order to gain insight into the structure and the dynamics of NPC1 lumenal domain for the cholesterol transport and the disease behind the mutation (R518W). It was found that the mutation induces a structural shift of the NTD (N-terminal domain), toward the loop region in the MLD (middle lumenal domain), which is believed to play a central role in the interaction with NPC2 protein, so the interaction with the NPC2 protein might be less favorable compared to the wild NPC1. Also, the simulation indicates the possible re-orientation of the NTD with both the wild and the R518W mutated NPC1 after receiving the cholesterol from the NPC2, that align to form an internal tunnel, which is a possible pose for further action in cholesterol trafficking. We believe the current study can provide a better understanding of the cholesterol transport by NPC1 especially the role of NTD of NPC1 in combination with NPC2 interactions.
    Keywords:  COVID‐19; NPC1; NPC2; R518W mutant; cholesterol trafficking; molecular dynamics simulation; neurodegenerative Niemann‐Pick C disease
    DOI:  https://doi.org/10.1111/jnc.15150
  11. Analyst. 2020 Sep 01.
    A red-emission fluorescent probe for visual monitoring of lysosomal pH changes during mitophagy and cell apoptosis.
    Xiaodong Wang, Li Fan, Xiaoran Zhang, Qi Zan, Wenjuan Dong, Shaomin Shuang, Chuan Dong.
      We presented a novel red-emission fluorescent probe (MSO) for selectively monitoring lysosomal pH fluctuation in living cells. The probe was designed by employing rhodamine B as the off-on pH sensitive moiety owing to the unique spirocycle group and morpholine as the lysosome targetable unit. Based on the H+-induced spirocyclic ring opening process, MSO displayed significant pH sensing properties around 590 nm, with a pKa value of 5.42 and a good linear pH response ranging from 5.00 to 6.00. Besides, the probe possessed other prominent photophysical properties such as good selectivity and excellent photostability as well as low cytotoxicity, together making the red-emission probe more favorable for long-time and real-time imaging in live cells. Furthermore, MSO selectively accumulated into lysosomes and successfully visualized the mitophagy, cell apoptosis and heat shock processes by monitoring the rise of lysosomal pH.
    DOI:  https://doi.org/10.1039/d0an01141j
  12. Gene Ther. 2020 Sep 03.
    Therapeutic benefit after intracranial gene therapy delivered during the symptomatic stage in a feline model of Sandhoff disease.
    Victoria J McCurdy, Aime K Johnson, Heather L Gray-Edwards, Ashley N Randle, Allison M Bradbury, Nancy E Morrison, Misako Hwang, Henry J Baker, Nancy R Cox, Miguel Sena-Esteves, Douglas R Martin.
      Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by defects in the β-subunit of β-N-acetylhexosaminidase (Hex), the enzyme that catabolizes GM2 ganglioside. Hex deficiency causes neuronal storage of GM2 and related glycoconjugates, resulting in progressive neurodegeneration and death, typically in infancy. No effective treatment exists for human patients. Adeno-associated virus (AAV) gene therapy led to improved clinical outcome and survival of SD cats treated before the onset of disease symptoms. Most human patients are diagnosed after clinical disease onset, so it is imperative to test AAV-gene therapy in symptomatic SD cats to provide a realistic indication of therapeutic benefits that can be expected in humans. In this study, AAVrh8 vectors injected into the thalamus and deep cerebellar nuclei of symptomatic SD cats resulted in widespread central nervous system enzyme distribution, although a substantial burden of storage material remained. Cats treated in the early symptomatic phase showed delayed disease progression and a significant survival increase versus untreated cats. Treatment was less effective when administered later in the disease course, although therapeutic benefit was still possible. Results are encouraging for the treatment of human patients and provide support for the development AAV-gene therapy for human SD.
    DOI:  https://doi.org/10.1038/s41434-020-00190-1
  13. FEBS Open Bio. 2020 Sep 03.
    Autophagy under glucose starvation enhances protein translation initiation in response to re-addition of glucose in C2C12 myotubes.
    Naoya Nakai, Saki Kitai, Noriko Iida, Sachika Inoue, Kazuhiko Higashida.
      Proteolysis is known to play a crucial role in maintaining skeletal muscle mass and function. Autophagy is a conserved intracellular process for the bulk degradation of proteins in lysosomes. Although nutrient starvation is known to induce autophagy, the effect of nutrient repletion following starvation on the mTOR pathway-mediated protein translation remains unclear. In the present study, we examined the effect of glucose starvation on the initiation of protein translation in response to glucose re-addition in C2C12 myotubes. Glucose starvation decreased the phosphorylation of p70 S6 kinase (p70S6K), a bonafide marker for protein translation initiation. Following re-addition of glucose, phosphorylation of p70S6K markedly increased only in glucose-starved cells. Inhibiting autophagy using pharmacological inhibitors diminished the effect of glucose re-addition on the phosphorylation of p70S6K, whereas inhibition of the ubiquitin-proteasome system did not exert any effect. In conclusion, autophagy under glucose starvation partially accounts for the activation of translation initiation by re-addition of glucose.
    Keywords:  autophagy; glucose starvation; mTORC1; p70 S6 kinase; protein synthesis
    DOI:  https://doi.org/10.1002/2211-5463.12970
  14. Cells. 2020 Sep 02. pii: E2022. [Epub ahead of print]9(9):
    Ubiquitin, Autophagy and Neurodegenerative Diseases.
    Yoshihisa Watanabe, Katsutoshi Taguchi, Masaki Tanaka.
      Ubiquitin signals play various roles in proteolytic and non-proteolytic functions. Ubiquitin signals are recognized as targets of the ubiquitin-proteasome system and the autophagy-lysosome pathway. In autophagy, ubiquitin signals are required for selective incorporation of cargoes, such as proteins, organelles, and microbial invaders, into autophagosomes. Autophagy receptors possessing an LC3-binding domain and a ubiquitin binding domain are involved in this process. Autophagy activity can decline as a result of genetic variation, aging, or lifestyle, resulting in the onset of various neurodegenerative diseases. This review summarizes the selective autophagy of neurodegenerative disease-associated protein aggregates via autophagy receptors and discusses its therapeutic application for neurodegenerative diseases.
    Keywords:  autophagy; autophagy–lysosome pathway; neurodegenerative diseases; ubiquitin; ubiquitin–proteasome system
    DOI:  https://doi.org/10.3390/cells9092022
  15. Proc Natl Acad Sci U S A. 2020 Sep 02. pii: 202008980. [Epub ahead of print]
    Decanoic acid inhibits mTORC1 activity independent of glucose and insulin signaling.
    Eleanor C Warren, Stephanie Dooves, Eleonora Lugarà, Joseph Damstra-Oddy, Judith Schaf, Vivi M Heine, Mathew C Walker, Robin S B Williams.
      Low-glucose and -insulin conditions, associated with ketogenic diets, can reduce the activity of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, potentially leading to a range of positive medical and health-related effects. Here, we determined whether mTORC1 signaling is also a target for decanoic acid, a key component of the medium-chain triglyceride (MCT) ketogenic diet. Using a tractable model system, Dictyostelium, we show that decanoic acid can decrease mTORC1 activity, under conditions of constant glucose and in the absence of insulin, measured by phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). We determine that this effect of decanoic acid is dependent on a ubiquitin regulatory X domain-containing protein, mediating inhibition of a conserved Dictyostelium AAA ATPase, p97, a homolog of the human transitional endoplasmic reticulum ATPase (VCP/p97) protein. We then demonstrate that decanoic acid decreases mTORC1 activity in the absence of insulin and under high-glucose conditions in ex vivo rat hippocampus and in tuberous sclerosis complex (TSC) patient-derived astrocytes. Our data therefore indicate that dietary decanoic acid may provide a new therapeutic approach to down-regulate mTORC1 signaling.
    Keywords:  Dictyostelium discoideum; decanoic acid; epilepsy; mTOR; tuberous sclerosis complex
    DOI:  https://doi.org/10.1073/pnas.2008980117
  16. EMBO J. 2020 Aug 31. e103791
    The Alzheimer's disease-associated C99 fragment of APP regulates cellular cholesterol trafficking.
    Jorge Montesinos, Marta Pera, Delfina Larrea, Cristina Guardia-Laguarta, Rishi R Agrawal, Kevin R Velasco, Taekyung D Yun, Irina G Stavrovskaya, Yimeng Xu, So Yeon Koo, Amanda M Snead, Andrew A Sproul, Estela Area-Gomez.
      The link between cholesterol homeostasis and cleavage of the amyloid precursor protein (APP), and how this relationship relates to Alzheimer's disease (AD) pathogenesis, is still unknown. Cellular cholesterol levels are regulated through crosstalk between the plasma membrane (PM), where most cellular cholesterol resides, and the endoplasmic reticulum (ER), where the protein machinery that regulates cholesterol levels resides. The intracellular transport of cholesterol from the PM to the ER is believed to be activated by a lipid-sensing peptide(s) in the ER that can cluster PM-derived cholesterol into transient detergent-resistant membrane domains (DRMs) within the ER, also called the ER regulatory pool of cholesterol. When formed, these cholesterol-rich domains in the ER maintain cellular homeostasis by inducing cholesterol esterification as a mechanism of detoxification while attenuating its de novo synthesis. In this manuscript, we propose that the 99-aa C-terminal fragment of APP (C99), when delivered to the ER for cleavage by γ-secretase, acts as a lipid-sensing peptide that forms regulatory DRMs in the ER, called mitochondria-associated ER membranes (MAM). Our data in cellular AD models indicates that increased levels of uncleaved C99 in the ER, an early phenotype of the disease, upregulates the formation of these transient DRMs by inducing the internalization of extracellular cholesterol and its trafficking from the PM to the ER. These results suggest a novel role for C99 as a mediator of cholesterol disturbances in AD, potentially explaining early hallmarks of the disease.
    Keywords:  Alzheimer's disease; amyloid precursor protein; cholesterol; lipid rafts; mitochondria-associated ER membranes
    DOI:  https://doi.org/10.15252/embj.2019103791
  17. Autophagy. 2020 Sep 02.
    The DNA methyltransferase DNMT3A contributes to autophagy long-term memory.
    Patricia González-Rodríguez, Mathilde Cheray, Jens Füllgrabe, Maria Salli, Pinelopi Engskog-Vlachos, Lily Keane, Virginia Cunha, Agata Lupa, Wenbo Li, Qi Ma, Kristian Dreij, Michael G Rosenfeld, Bertrand Joseph.
      Macroautophagy/autophagy is a conserved catabolic pathway that targets cytoplasmic components for their degradation and recycling in an autophagosome-dependent lysosomal manner. Under physiological conditions, this process maintains cellular homeostasis. However, autophagy can be stimulated upon different forms of cellular stress, ranging from nutrient starvation to exposure to drugs. Thus, this pathway can be seen as a central component of the integrated and adaptive stress response. Here, we report that even brief induction of autophagy is coupled in vitro to a persistent downregulation of the expression of MAP1LC3 isoforms, which are key components of the autophagy core machinery. In fact, DNA-methylation mediated by de novo DNA methyltransferase DNMT3A of MAP1LC3 loci upon autophagy stimulation leads to the observed long-term decrease of MAP1LC3 isoforms at transcriptional level. Finally, we report that the downregulation of MAP1LC3 expression can be observed in vivo in zebrafish larvae and mice exposed to a transient autophagy stimulus. This epigenetic memory of autophagy provides some understanding of the long-term effect of autophagy induction and offers a possible mechanism for its decline upon aging, pathological conditions, or in response to treatment interventions.
    Keywords:  Autophagy; DNA methylation; MAP1LC3; epigenetics; transcription
    DOI:  https://doi.org/10.1080/15548627.2020.1816664
  18. J Neurochem. 2020 Aug 31.
    Distinct alpha-Synuclein species induced by seeding are selectively cleared by the Lysosome or the Proteasome in neuronally differentiated SH-SY5Y cells.
    Marina Pantazopoulou, Viviana Brembati, Angeliki Kanellidi, Luc Bousset, Ronald Melki, Leonidas Stefanis.
      A major pathological feature of Parkinson's disease (PD) is the aberrant accumulation of misfolded assemblies of alpha-synuclein (α-Syn). Protein clearance appears as a regulator of the ''α-Syn burden" underlying PD pathogenesis. The picture emerging is that a combination of pathways with complementary roles, including the Proteasome System and the Autophagy-Lysosome Pathway, contributes to the intracellular degradation of α-Syn. The current study addresses the mechanisms governing the degradation of α-Syn species seeded by exogenous fibrils in neuronally differentiated SH-SY5Y neuroblastoma cells with inducible expression of α-Syn. Using human α-Syn recombinant fibrils (pre-formed fibrils, PFFs), seeding and aggregation of endogenous Proteinase K (PK)-resistant α-Syn species occurs within a time frame of 6 days, and is still prominent after 12 days of PFF addition. Clearance of α-Syn assemblies in this inducible model was enhanced after switching off α-Syn expression with doxycycline. Lysosomal inhibition led to accumulation of SDS-soluble α-Syn aggregates 6 days after PFF-addition or when switching off α-Syn expression. Additionally, the autophagic enhancer, rapamycin, induced the clearance of α-Syn aggregates 13 days post-PFF addition, indicating that autophagy is the major pathway for aggregated α-Syn clearance. SDS-soluble phosphorylated α-Syn at S129 was only apparent at 7 days of incubation with a higher amount of PFFs. Proteasomal inhibition resulted in further accumulation of SDS-soluble phosphorylated α-Syn at S129, with limited PK resistance. Our data suggest that in this inducible model autophagy is mainly responsible for the degradation of fibrillar α-Syn, whereas the Proteasome System is responsible, at least in part, for the selective clearance of phosphorylated α-Syn oligomers.
    Keywords:  aggregation; alpha-synuclein; degradation; lysosome; phosphorylation; proteasome
    DOI:  https://doi.org/10.1111/jnc.15174
  19. J Biol Chem. 2020 Sep 02. pii: jbc.RA120.013121. [Epub ahead of print]
    Branched-chain ketoacid overload inhibits insulin action in the muscle.
    Dipsikha Biswas, Khoi T Dao, Angella Mercer, Andrew M Cowie, Luke Duffley, Yassine El Hiani, Petra C Kienesberger, Thomas Pulinilkunnil.
      Branched-chain α-keto acids (BCKAs) are catabolites of branched-chain amino acids (BCAAs). Intracellular BCKAs is cleared by branched-chain ketoacid dehydrogenase (BCKDH), which is sensitive to inhibitory phosphorylation by BCKD kinase (BCKDK). Accumulation of BCKAs is an indicator of defective BCAA catabolism and has been correlated with glucose intolerance and cardiac dysfunction. However, it is unclear whether BCKAs directly alter insulin signaling and function in the skeletal and cardiac muscle cell. Furthermore, the role of excess fatty acids (FA) in perturbing BCAA catabolism and BCKA availability merits investigation. By using immunoblot and UPLC MS/MS to analyze the hearts of fasted mice, we observed decreased BCAA catabolizing enzyme expression and increased circulating BCKAs, but not BCAAs. In mice subjected to diet-induced obesity (DIO), we observed similar increases in circulating BCKAs with concomitant changes in BCAA catabolizing enzyme expression only in the skeletal muscle. Effects of DIO were recapitulated by simulating lipotoxicity in skeletal muscle cells treated with saturated FA, palmitate. Exposure of muscle cells to high concentrations of BCKAs resulted in inhibition of insulin-induced AKT phosphorylation, decreased glucose uptake and mitochondrial oxygen consumption. Altering intracellular clearance of BCKAs by genetic modulation of BCKDK and BCKDHA expression showed similar effects on AKT phosphorylation. BCKAs increased protein translation and mTORC1 activation. Pretreating cells with mTORC1 inhibitor rapamycin restored BCKAs effect on insulin-induced AKT phosphorylation. This study provides evidence for FA mediated regulation of BCAA catabolizing enzymes, BCKA content and highlights the biological role of BCKAs in regulating muscle insulin signaling and function.
    Keywords:  BCKA; Insulin signaling; amino acid; cardiomyocyte; cardiomyocytes; insulin resistance; protein translation; skeletal muscle; skeletal muscle metabolism; translation
    DOI:  https://doi.org/10.1074/jbc.RA120.013121
  20. Autophagy. 2020 Sep 02.
    PDPK1 regulates autophagosome biogenesis by binding to PIK3C3.
    Boli Hu, Yina Zhang, Tingjuan Deng, Jinyan Gu, Juan Liu, Hui Yang, Yuting Xu, Yan Yan, Fan Yang, Heng Zhang, Yulan Jin, Jiyong Zhou.
      PDPK1 (3-phosphoinositide dependent protein kinase 1) is a phosphorylation-regulated kinase that plays a central role in activating multiple signaling pathways and cellular processes. Here, this study shows that PDPK1 turns on macroautophagy/autophagy as a SUMOylation-regulated kinase. In vivo data demonstrate that the SUMO modification of PDPK1 is a physiological feature in the brain and that it can be induced by viral infections. The SUMOylated PDPK1 regulates its own phosphorylation and subsequent activation of the AKT1 (AKT serine/threonine kinase 1)-MTOR (mechanistic target of rapamycin kinase) pathway. However, SUMOylation of PDPK1 is inhibited by binding to PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3). The nonSUMOylated PDPK1 then tethers LC3 to the endoplasmic reticulum to initiate autophagy, and it acts as a key component in forming the autophagic vacuole. Collectively, this study reveals the intricate molecular regulation of PDPK1 by post-translational modification in controlling autophagosome biogenesis, and it highlights the role of PDPK1 as a sensor of cellular stress and regulator of autophagosome biogenesis.
    Keywords:  AKT1-MTOR; PDPK1; PIK3C3; SUMOylation; autophagy
    DOI:  https://doi.org/10.1080/15548627.2020.1817279
  21. J Infus Nurs. 2020 Sep/Oct;43(5):43(5): 243-245
    Enzyme Replacement Therapy for Lysosomal Storage Diseases.
    Susan Kleppin.
      
    DOI:  https://doi.org/10.1097/NAN.0000000000000390
  22. Biochem J. 2020 Sep 04. pii: BCJ20200551. [Epub ahead of print]
    Amino acid starvation inhibits autophagy in lipid droplet-deficient cells through mitochondrial dysfunction.
    Pierre Voisin, Marianne Bernard, Thierry Bergès, Matthieu Regnacq.
      Lipid droplets are ubiquitous organelles in eukaryotes that act as storage sites for neutral lipids. Under normal growth conditions they are not required in the yeast Saccharomyces cerevisiae. However, recent works have shown that lipid droplets are required for autophagy to proceed in response to nitrogen starvation and that they play an essential role in maintaining ER homeostasis. Autophagy is a major catabolic pathway that helps degradation and recycling of potentially harmful proteins and organelles. It can be pharmacologically induced by rapamycin even in the absence of lipid droplets. Here, we show that amino acid starvation is responsible for autophagy failure in lipid droplet-deficient yeast.  It not only fails to induce autophagy but also inhibits rapamycin-induced autophagy. The general amino acid control pathway is not involved in this paradoxical effect of amino acid shortage. We correlate the autophagy failure with mitochondria aggregation and we show that amino acid starvation-induced autophagy is restored in lipid droplet-deficient yeast by increasing mitochondrial biomass physiologically (respiration) or genetically (REG1 deletion). Our results establish a new functional link between lipid droplets, ER and mitochondria during nitrogen starvation-induced autophagy.
    Keywords:  Autophagy; Saccharomyces cerevisiae; catabolite repression; lipid droplets; mitochondrial dysfunction
    DOI:  https://doi.org/10.1042/BCJ20200551
  23. Metab Brain Dis. 2020 Sep 04.
    Changes in expressions of genes involved in the regulation of cellular processes in mucopolysaccharidoses as assessed by fibroblast culture-based transcriptomic analyses.
    Lidia Gaffke, Karolina Pierzynowska, Karolina Krzelowska, Ewa Piotrowska, Grzegorz Węgrzyn.
      Recent studies indicated that apart from lysosomal storage of glycosaminoglycans (GAGs), secondary and tertiary changes in cellular processes may significantly contribute to development of disorders and symptoms occurring in mucopolysaccharidoses (MPS), a group of lysosomal storage diseases in which neurodegeneration is specific for most types and subtypes. In this report, using transcriptomic data, we demonstrate that regulation of hundreds of genes coding for proteins involved in regulations of various cellular processes is changed in cells derived from patients suffering from all types and subtypes of MPS. Among such genes there are 10 which expression is significantly changed in 9 or more (out of 11) MPS types/subtypes; they include IER3IP1, SAR1A, TMEM38B, PLCB4, SIN3B, ABHD5, SH3BP5, CAPG, PCOLCE2, and MN1. Moreover, there are several genes whose expression is changed over log2 > 4 times in some MPS types relative to control cells. The above analysis indicates that significant changes in expression of genes coding for various regulators of cellular processes may considerably contribute to development of cellular dysfunctions, and further appearance of specific symptoms of MPS, including neurodegeneration.
    Keywords:  Mucopolysaccharidoses; Regulation of cellular processes; Transcriptomics
    DOI:  https://doi.org/10.1007/s11011-020-00614-2
  24. Chem Biol Interact. 2020 Aug 27. pii: S0009-2797(19)31503-0. [Epub ahead of print] 109243
    Upregulation of Akt/Raptor signaling is associated with rapamycin resistance of breast cancer cells.
    Yuri Shchegolev, Danila Sorokin, Alexander Scherbakov, Alexey Shunaev, Olga Andreeva, Ekaterina Mikhaevich, Margarita Gudkova, Irina Bure, Lev Berstein, Marina Nemtsova, Mikhail Krasil'nikov.
      mTOR inhibitors are considered today to be one of the most promising anticancer drugs. Here to study the mechanism of the acquired resistance of MCF-7 breast cancer cells to mTOR inhibitors two different models of the cell resistance were used: rapamycin-resistant MCF-7/Rap subline developed under long-term rapamycin treatment, and metformin-resistant MCF-7/M subline obtained by long-term metformin treatment. We have found that both resistant sublines were characterized by common features: increased expression of mTOR-interacting Raptor protein, increased phosphorylation of Akt, and activation of growth-related transcriptional factor AP-1. Cell response to mTOR inhibitors was partially restored under treatment with PI3K inhibitor wortmannin supporting the direct connection between Akt activation and poor cell response to therapeutic drugs. Transfection of mir-181c, one of the positive regulators of Akt and mTOR, lead to an increase in the cell resistance to both mTOR inhibitors, rapamycin, and metformin, which correlated with Raptor overexpression and activation of Akt/AP-1 signaling. In general, the effect of Raptor overexpression in the resistant cells, as well as the ability of mir-181c to modulate the Raptor expression, can open novel perspectives in the treatment of rapalogues-resistant cancers, based on the drugs design targeting mir-181c/Raptor axis.
    Keywords:  Breast cancer; Metformin; Rapamycin; Raptor; mTOR; miR-181c
    DOI:  https://doi.org/10.1016/j.cbi.2020.109243
  25. Mol Genet Metab. 2020 Aug 28. pii: S1096-7192(20)30187-6. [Epub ahead of print]
    Detailed epitope mapping of neutralizing anti-drug antibodies against recombinant α-galactosidase A in patients with Fabry disease.
    David Scharnetzki, Franciska Stappers, Malte Lenders, Eva Brand.
      BACKGROUND: Fabry disease (FD) is a lysosomal storage disease, treatable by enzyme replacement therapy (ERT) that substitutes deficient α-galactosidase A (AGAL). The formation of neutralizing anti-drug antibodies (ADA) inhibiting AGAL activity is associated with disease progression in affected male patients. In the current study, we performed a detailed epitope mapping of ADAs from antibody-positive males against infused AGAL.METHODS: A detailed epitope mapping for 34 male FD patients with neutralizing ADAs against AGAL was performed. Based on this data, in silico analyses were used to identify potential epitope clusters and mapped surface-located or buried epitopes. ELISA-based assays against α-galactosidase B (NAGA) were performed to identify ADAs that potentially recognize shared epitopes of AGAL and NAGA. A subset of 20 patients was analyzed to assess if NAGA-recognizing ADAs against AGAL might affect long-term outcomes under ERT.
    RESULTS: Thirty percent of the AGAL active site was recognized by patients' ADAs. No differences between buried and surface-located epitopes were observed. Dependent on the epitopes, ADAs against AGAL were also able to recognize human NAGA. Patients with NAGA recognizing anti-AGAL antibodies presented with lower plasma NAGA activities. The presence of NAGA-recognizing ADAs had no effect on disease progression.
    CONCLUSION: In conclusion, our current data underline previous reports demonstrating a large variation of antibody epitopes against AGAL. Detailed epitope mapping in affected patients might be the first step for the generation of patient-specific blocking peptides and/or immune adsorption columns for an individually tailored anti-antibody strategy.
    Keywords:  Anti-drug antibodies; Enzyme replacement therapy; Epitopes; Fabry disease; Globotriaosylceramide
    DOI:  https://doi.org/10.1016/j.ymgme.2020.08.005
  26. Rev Prat. 2020 Apr;70(4): 416-420
    [Gaucher disease].
    Yann Nguyen, Jérôme Stirnemann, Nadia Belmatoug.
      Gaucher disease. Gaucher disease is a rare lysosomal autosomal recessive disease, caused by a deficiency of glucocerebrosidase, a lysosomal enzyme. The most frequent symptoms are cytopenia, splenomegaly, hepatomegaly, and potentially severe bone involvement (bone infarcts, avascular osteonecrosis, and pathological fractures). Neurological involvement may occur in type 2 and type 3 Gaucher disease. Patients with type 1 Gaucher disease have an increased risk of Parkinson disease, some solid cancers, and some hematologic malignancies including multiple myeloma. Patients often experience delays before their disease is being diagnosed. Thus, there is a need for physicians to recognize Gaucher disease symptoms to reduce the risk of irreversible complications.
  27. Cells. 2020 Sep 02. pii: E2025. [Epub ahead of print]9(9):
    The Roles of Ubiquitin in Mediating Autophagy.
    Zhangyuan Yin, Hana Popelka, Yuchen Lei, Ying Yang, Daniel J Klionsky.
      Ubiquitination, the post-translational modification essential for various intracellular processes, is implicated in multiple aspects of autophagy, the major lysosome/vacuole-dependent degradation pathway. The autophagy machinery adopted the structural architecture of ubiquitin and employs two ubiquitin-like protein conjugation systems for autophagosome biogenesis. Ubiquitin chains that are attached as labels to protein aggregates or subcellular organelles confer selectivity, allowing autophagy receptors to simultaneously bind ubiquitinated cargos and autophagy-specific ubiquitin-like modifiers (Atg8-family proteins). Moreover, there is tremendous crosstalk between autophagy and the ubiquitin-proteasome system. Ubiquitination of autophagy-related proteins or regulatory components plays significant roles in the precise control of the autophagy pathway. In this review, we summarize and discuss the molecular mechanisms and functions of ubiquitin and ubiquitination, in the process and regulation of autophagy.
    Keywords:  autophagy; lysosome; selective autophagy; ubiquitin; ubiquitination
    DOI:  https://doi.org/10.3390/cells9092025
  28. J Biol Chem. 2020 Sep 02. pii: jbc.RA120.012992. [Epub ahead of print]
    C-terminal tail length guides insertion and assembly of membrane proteins.
    Sha Sun, Malaiyalam Mariappam.
      A large number of newly synthesized membrane proteins in the endoplasmic reticulum (ER) are assembled into multi-protein complexes, but little is known about the mechanisms required for assembly membrane proteins. It has been suggested that membrane chaperones might exist, akin to the molecular chaperones that stabilize and direct the assembly of soluble protein complexes, but the mechanisms by which these proteins would bring together membrane protein components is unclear. Here, we have identified that the tail length of the C-terminal transmembrane domains (C-TMDs) determines efficient insertion and assembly of membrane proteins in the ER. We found that membrane proteins with C-TMD tails shorter than ~60 amino acids are poorly inserted into the ER membrane, which suggests that translation is terminated before they are recognized by the Sec61 translocon for insertion. These C-TMDs with insufficient hydrophobicity are post-translationally recognized and retained by the Sec61 translocon complex, providing a time window for efficient assembly with TMDs from partner proteins. Retained TMDs that fail to assemble with their cognate TMDs are slowly translocated into the ER lumen and are recognized by the ER-associated degradation (ERAD) pathway for removal. In contrast, C-TMDs with sufficient hydrophobicity or tails longer than ~80 residues are quickly released from the Sec61 translocon into the membrane or the ER lumen, resulting in inefficient assembly with partner TMDs. Thus, our data suggest that C-terminal tails harbor crucial signals for both the insertion and assembly of membrane proteins.
    Keywords:  endoplasmic reticulum (ER); membrane protein; protein assembly; protein degradation; translocation
    DOI:  https://doi.org/10.1074/jbc.RA120.012992
  29. Redox Biol. 2020 Jul 09. pii: S2213-2317(20)30842-9. [Epub ahead of print]36 101637
    Substrate metabolism regulated by Sestrin2-mTORC1 alleviates pressure overload-induced cardiac hypertrophy in aged heart.
    Nanhu Quan, Xuan Li, Jingwen Zhang, Ying Han, Weiju Sun, Di Ren, Qian Tong, Ji Li.
      Sestrin2 (Sesn2) is a stress sensor for the mammalian target of rapamycin complex 1 (mTORC1) pathway. Aging impairs cardiac mTORC1 activation, thereby sensitizing the heart to hypertrophy. C57BL/6 J young wild-type (young WT; 4-6 months), aged WT (24-26 months), and young Sestrin2 knockout mice (Y-Sesn2 KO; 4-6 months) underwent transverse aortic constriction (TAC) for pressure overload. Cardiac expression of Sesn2 decreased with age. At 4 weeks after TAC, aged WT and Y-Sesn2 KO exhibited larger hearts and impaired cardiac function, compared with young WT mice. Augmented phosphorylation of mTOR and downstream effectors; damaged mitochondria and elevated redox markers, as well as and impaired glucose and fatty acid oxidation were observed in aged WT and Y-Sesn2 KO hearts. A pressure overload-induced interaction between Sesn2 and GTPase-activating protein activity toward Rags 2 (GATOR2), which positively regulates mTORC1, was impaired in aged WT hearts. Adeno-associated virus 9-Sesn2 treatment rescued Sesn2 expression, attenuated mTORC1 activation, and increased pressure overload tolerance in aged WT and Y-Sesn2 KO hearts. These results indicated that cardiac Sesn2 acts as a pressure overload sensor for mTORC1. Furthermore, Sesn2 deficiency may cause increased sensitivity to hypertrophy in elderly individuals.
    Keywords:  Aging; Hypertrophy; Pressure overload; Sestrin2; mTORC1
    DOI:  https://doi.org/10.1016/j.redox.2020.101637
  30. Mol Neurobiol. 2020 Aug 31.
    Antioxidant Modulation of mTOR and Sirtuin Pathways in Age-Related Neurodegenerative Diseases.
    Asmaa Abdullah, Nuraqila Mohd Murshid, Suzana Makpol.
      In the human body, cell division and metabolism are expected to transpire uneventfully for approximately 25 years. Then, secondary metabolism and cell damage products accumulate, and ageing phenotypes are acquired, causing the progression of disease. Among these age-related diseases, neurodegenerative diseases have attracted considerable attention because of their irreversibility, the absence of effective treatment and their relationship with social and economic pressures. Mechanistic (formerly mammalian) target of rapamycin (mTOR), sirtuin (SIRT) and insulin/insulin growth factor 1 (IGF1) signalling pathways are among the most important pathways in ageing-associated conditions, such as neurodegeneration. These longevity-related pathways are associated with a diversity of various processes, including metabolism, cognition, stress reaction and brain plasticity. In this review, we discuss the roles of sirtuin and mTOR in ageing and neurodegeneration, with an emphasis on their regulation of autophagy, apoptosis and mitochondrial energy metabolism. The intervention of neurodegeneration using potential antioxidants, including vitamins, phytochemicals, resveratrol, herbals, curcumin, coenzyme Q10 and minerals, specifically aimed at retaining mitochondrial function in the treatment of Alzheimer's disease, Parkinson's disease and Huntington's disease is highlighted.
    Keywords:  Ageing; Antioxidants; Neurodegenerative diseases; Sirtuins; mTOR
    DOI:  https://doi.org/10.1007/s12035-020-02083-1
  31. PLoS One. 2020 ;15(9): e0238546
    MITF and TFEB cross-regulation in melanoma cells.
    Josué Ballesteros-Álvarez, Ramile Dilshat, Valerie Fock, Katrín Möller, Ludwig Karl, Lionel Larue, Margrét Helga Ögmundsdóttir, Eiríkur Steingrímsson.
      The MITF, TFEB, TFE3 and TFEC (MiT-TFE) proteins belong to the basic helix-loop-helix family of leucine zipper transcription factors. MITF is crucial for melanocyte development and differentiation, and has been termed a lineage-specific oncogene in melanoma. The three related proteins MITF, TFEB and TFE3 have been shown to be involved in the biogenesis and function of lysosomes and autophagosomes, regulating cellular clearance pathways. Here we investigated the cross-regulatory relationship of MITF and TFEB in melanoma cells. Like MITF, the TFEB and TFE3 genes are expressed in melanoma cells as well as in melanoma tumors, albeit at lower levels. We show that the MITF and TFEB proteins, but not TFE3, directly affect each other's mRNA and protein expression. In addition, the subcellular localization of MITF and TFEB is subject to regulation by the mTOR signaling pathway, which impacts their cross-regulatory relationship at the transcriptional level. Our work shows that the relationship between MITF and TFEB is multifaceted and that the cross-regulatory interactions of these factors need to be taken into account when considering pathways regulated by these proteins.
    DOI:  https://doi.org/10.1371/journal.pone.0238546
  32. Science. 2020 Sep 04. pii: eaaz7714. [Epub ahead of print]369(6508):
    Reconstitution of autophagosome nucleation defines Atg9 vesicles as seeds for membrane formation.
    Justyna Sawa-Makarska, Verena Baumann, Nicolas Coudevylle, Sören von Bülow, Veronika Nogellova, Christine Abert, Martina Schuschnig, Martin Graef, Gerhard Hummer, Sascha Martens.
      Autophagosomes form de novo in a manner that is incompletely understood. Particularly enigmatic are autophagy-related protein 9 (Atg9)-containing vesicles that are required for autophagy machinery assembly but do not supply the bulk of the autophagosomal membrane. In this study, we reconstituted autophagosome nucleation using recombinant components from yeast. We found that Atg9 proteoliposomes first recruited the phosphatidylinositol 3-phosphate kinase complex, followed by Atg21, the Atg2-Atg18 lipid transfer complex, and the E3-like Atg12-Atg5-Atg16 complex, which promoted Atg8 lipidation. Furthermore, we found that Atg2 could transfer lipids for Atg8 lipidation. In selective autophagy, these reactions could potentially be coupled to the cargo via the Atg19-Atg11-Atg9 interactions. We thus propose that Atg9 vesicles form seeds that establish membrane contact sites to initiate lipid transfer from compartments such as the endoplasmic reticulum.
    DOI:  https://doi.org/10.1126/science.aaz7714
  33. BMC Ophthalmol. 2020 Aug 31. 20(1): 356
    Bergmeister's papilla in a young patient with type 1 sialidosis: case report.
    Settimio Rossi, Carlo Gesualdo, Antonio Tartaglione, Leonilda Bilo, Antonietta Coppola, Francesca Simonelli.
      BACKGROUND: Sialidosis is a rare genetic lysosomal storage disorder caused by a deficit of neuraminidase enzyme activity. Patients with sialidosis present various neurological disorders such as: myoclonic epilepsy and hypotonia, often associated with visual impairment. A typical aspect of sialidosis is the finding of a macular cherry-red spot on ocular fundus examination. In this paper we describe a unilateral case of Bergmeister's papilla (BP) in a young female patient suffering from type 1 sialidosis.CASE PRESENTATION: A 28-year-old young woman suffering from type 1 sialidosis, confirmed by previously described compound heterozigosity Leu91Arg and Gly328Ser on N-acetyl-alpha-neuraminidase - 1 (NEU1) gene, underwent an opthalmological examination at the Eye Clinic of the University of Campania L. Vanvitelli, for bilateral visual deterioration. The patient was suffering from myoclonic epilepsy with hypotonia and severe motor disability. Fundoscopic examination showed a typical macular cherry-red spot with retinal pigment epithelium dystrophy in the middle periphery, in both eyes. Furthermore, in the left eye (OS), a vitreous thickening was observed in the nasal sector of the optic disc, remnant of fetal vasculature on the optic disc (Bergmeister's papilla). Optical coherence tomography (OCT) showed, in both eyes, a thickening of the ganglion cell layer (GCL) with a hyperreflective opacity as a cap on the left optic disc.
    CONCLUSIONS: In our paper we have described, for the first time in literature, a case of BP in a patient with type 1 sialidosis. The detection of BP with thickening of the peripapillary vitreous by SD-OCT is useful in monitoring any vitreo-retinal change that could cause future visual deterioration.
    Keywords:  Bergmeister’s papilla; Optical coherence tomography; Sialidosis
    DOI:  https://doi.org/10.1186/s12886-020-01628-1
  34. Elife. 2020 Sep 02. pii: e58737. [Epub ahead of print]9
    mTOR signaling regulates the morphology and migration of outer radial glia in developing human cortex.
    Madeline G Andrews, Lakshmi Subramanian, Arnold R Kriegstein.
      Outer radial glial (oRG) cells are a population of neural stem cells prevalent in the developing human cortex that contribute to its cellular diversity and evolutionary expansion. The mammalian Target of Rapamycin (mTOR) signaling pathway is active in human oRG cells. Mutations in mTOR pathway genes are linked to a variety of neurodevelopmental disorders and malformations of cortical development. We find that dysregulation of mTOR signaling specifically affects oRG cells, but not other progenitor types, by changing the actin cytoskeleton through the activity of the Rho-GTPase, CDC42. These effects change oRG cellular morphology, migration, and mitotic behavior, but do not affect proliferation or cell fate. Thus, mTOR signaling can regulate the architecture of the developing human cortex by maintaining the cytoskeletal organization of oRG cells and the radial glia scaffold. Our study provides insight into how mTOR dysregulation may contribute to neurodevelopmental disease.
    Keywords:  human; human cortex; neuroscience; organoids; outer radial glia; regenerative medicine; stem cells
    DOI:  https://doi.org/10.7554/eLife.58737
  35. Mol Genet Genomic Med. 2020 Sep 01. e1478
    Identification of a missense ARSA mutation in metachromatic leukodystrophy and its potential pathogenic mechanism.
    Liyuan Guo, Bo Jin, Yidan Zhang, Jing Wang.
      BACKGROUND: Metachromatic leukodystrophy (MLD) is a rare inherited lysosomal disorder caused by mutations in ARSA. The biological processes of MLD disease caused by candidate pathogenic mutations in the ARSA gene remain unclear.METHODS: We used whole-exome sequencing (WES) and Sanger sequencing to identify the pathogenic mutation in a Chinese family. Literature review and protein three-dimensional structure prediction were performed to analyze the potential pathogenesis of the identified mutations. Overexpression cell models of wild-type and mutated ARSA genes were constructed. The accumulated sulfatides and expression profiles in the cell models were detected, and a series of bioinformatics analyses were carried out to compare the biological changes caused by the candidate pathogenic mutations.
    RESULTS: We identified an ARSA c.925G>A homozygous mutation from a Chinese late-infantile MLD patient, the first report of this mutation in East Asia. The literature and protein structure analysis indicated that three types of mutations at c.925G (c.925G>A, c.925G>T, c.925G>C) were pathogenic. The overexpression of wild-type or mutated ARSA genes influenced the accumulation of sulfatides. The co-expression modules in the mutated cell models were constructed by genes related to calcium signaling and vesicle transport.
    CONCLUSION: Our results identified a pathogenic mutation, ARSA homozygosity c.925G>A, from a Chinese MLD family. The pathogenic mechanism of the ARSA mutation in MLD was identified, which may suggest new approaches to diagnosis and treatment.
    DOI:  https://doi.org/10.1002/mgg3.1478
  36. Mol Neurobiol. 2020 Sep 02.
    An Experimental Model of Neurodegenerative Disease Based on Porcine Hemagglutinating Encephalomyelitis Virus-Related Lysosomal Abnormalities.
    Yungang Lan, Zi Li, Zhenzhen Wang, Xinran Wang, Gaili Wang, Jing Zhang, Shiyu Hu, Kui Zhao, Baofeng Xu, Feng Gao, Wenqi He.
      Lysosomes are involved in pathogenesis of a variety of neurodegenerative diseases and play a large role in neurodegenerative disorders caused by virus infection. However, whether virus-infected cells or animals can be used as experimental models of neurodegeneration in humans based on virus-related lysosomal dysfunction remain unclear. Porcine hemagglutinating encephalomyelitis virus displays neurotropism in mice, and neural cells are its targets for viral progression. PHEV infection was confirmed to be a risk factor for neurodegenerative diseases in the present. The findings demonstrated for the first time that PHEV infection can lead to lysosome disorders and showed that the specific mechanism of lysosome dysfunction is related to PGRN expression deficiency and indicated similar pathogenesis compared with human neurodegenerative diseases upon PHEV infection. Trehalose can also increase progranulin expression and rescue abnormalities in lysosomal structure in PHEV-infected cells. In conclusion, these results suggest that PHEV probably serve as a disease model for studying the pathogenic mechanisms and prevention of other degenerative diseases.
    Keywords:  Lysosomal abnormalities; Neurodegenerative diseases; Porcine hemagglutinating encephalomyelitis virus; Progranulin; Trehalose
    DOI:  https://doi.org/10.1007/s12035-020-02105-y
  37. Autophagy. 2020 Aug 31.
    Epr1, a UPR-upregulated soluble autophagy receptor for reticulophagy.
    Dan Zhao, Li-Lin Du.
      The endoplasmic reticulum (ER) is a major site of protein folding. Perturbations in the folding capacity of the ER result in ER stress. ER stress triggers autophagic degradation of the ER (reticulophagy). Molecular mechanisms underlying ER stress-induced reticulophagy remain largely unknown. Our recent study identified a soluble protein, Epr1, as an autophagy receptor for ER stress-induced reticulophagy in the fission yeast Schizosaccharomyces pombe. Epr1 can interact simultaneously with Atg8 and a VAP family integral ER membrane protein, and thereby act as a bridging molecule between them. VAP family proteins contribute to reticulophagy by not only connecting Atg8 to the ER membrane through Epr1, but also by supporting the ER-plasma membrane contact. The expression of Epr1 is upregulated during ER stress in a manner dependent on the unfolded protein response (UPR) regulator Ire1. Ire1 promotes reticulophagy by upregulating Epr1.
    Keywords:  ER stress; ER-phagy; ER-plasma membrane contact; Ire1; VAP; reticulophagy; selective autophagy; unfolded protein response (UPR)
    DOI:  https://doi.org/10.1080/15548627.2020.1816665
  38. Mol Psychiatry. 2020 Sep 01.
    Reconsolidation of a post-ingestive nutrient memory requires mTOR in the central amygdala.
    Yuhua Yan, Lingli Zhang, Tailin Zhu, Shining Deng, Bingke Ma, Hui Lv, Xingyue Shan, Haidi Cheng, Kangli Jiang, Tiantian Zhang, Bo Meng, Bing Mei, Wei-Guang Li, Fei Li.
      The central control of feeding behavior and metabolic homeostasis has been proposed to involve a form of post-ingestive nutrient learning independent of the gustatory value of food. However, after such learning, it is unknown which brain regions or circuits are activated to retrieve the stored memory and whether this memory undergoes reconsolidation that depends on protein synthesis after its reactivation through retrieval. In the present study, using a conditioned-flavor-preference paradigm by associating flavors with intra-gastric infusion of glucose to minimize the evaluation of the taste of food, we show that retrieval of the post-ingestive nutrient-conditioned flavor memory stimulates multiple brain regions in mice, including the central nucleus of the amygdala (CeA). Moreover, memory retrieval activated the mammalian target of rapamycin complex 1 (mTORC1) in the CeA, while site-specific or systemic inhibition of mTORC1 immediately after retrieval prevented the subsequent expression of the post-ingestive nutrient-associated flavor memory, leading to a long-lasting suppression of reinstatement. Taken together, our findings suggest that the reconsolidation process of a post-ingestive nutrient memory modulates food preferences.
    DOI:  https://doi.org/10.1038/s41380-020-00874-5
  39. CNS Neurol Disord Drug Targets. 2020 Aug 31.
    Minocycline inhibits mTOR signaling activation and alleviates behavioral deficits in the Wistar rats with acute ischemia stroke.
    Shengyuan Wang, Chuanling Wang, Lihua Wang, Zhiyou Cai.
      BACKGROUND: Mammalian target of rapamycin (mTOR) has been evidenced as a multimodal therapy in the path-ophysiological process of acute ischemic stroke (AIS). However, the pathway that minocycline targets mTOR signaling is not fully defined in the AIS pathogenesis. This study is to aim at the effects of minocycline on the mTOR signaling in the AIS process and further discover the underlying mechanisms of minocycline involved in the following change of mTOR signaling-autophagy.METHODS: Cerebral ischemia/reperfusion (CIR) rat animal models were established with the transient suture occlusion into middle cerebral artery. Minocycline (50mg/kg) was given by intragastric administration. The Morris water maze was used to test the cognitive function of animals. Immunohisto chemistry and immuno fluorescence were introduced for testing the lev-els of synaptophysin and PSD-95. Western blot was conducted for investigating the levels of mTOR, p-mTOR (Ser2448), p70S6, p-p70S6 (Thr389), eEF2k, p-eEF2k (Ser366), p-eIF4B (Ser406), LC3, p62, synaptophysin and PSD-95.
    RESULTS: Minocycline prevents cognitive decline of the MCAO stroke rats. Minocycline limits the expression of p-mTOR (Ser2448) and the downstream targets of mTOR [p70S6, p-p70S6 (Thr389), eEF2k, p-eEF2k (Ser366) and p-eIF4B (Ser406)] (P<0.01), while minocycline has no influence on mTOR. LC3-II abundance and the LC3-II/I ratio were upregu-lated in the hippocampus of the MCAO stroke rats by the minocycline therapy (P<0.01). p62 was downregulated in the hip-pocampus from the MCAO stroke rats administrated with minocycline therapy(P<0.01). The levels of SYP and PSD-95 were up-regulated in the brain of the MCAO stroke rats administrated with minocycline therapy.
    CONCLUSION: Minocycline prevents cognitive deficits via inhibiting mTOR signaling and enhancing autophagy process, and promoting the expression of pre-and postsynaptic proteins (synaptophysin and PSD-95) in the brain of the MCAO stroke rats. The potential neuroprotective role of minocycline in the process of cerebral ischemia may be related to mitigating is-chemia-induced synapse injury via inhibiting activation of mTOR signaling.
    Keywords:  Minocycline; autophagy; behavioral deficits; cerebral ischemia; mammalian target of rapamycin; synapse
    DOI:  https://doi.org/10.2174/1871527319999200831153748
  40. Cells. 2020 Sep 01. pii: E2008. [Epub ahead of print]9(9):
    Atg8-Family Proteins-Structural Features and Molecular Interactions in Autophagy and Beyond.
    Nicole Wesch, Vladimir Kirkin, Vladimir V Rogov.
      Autophagy is a common name for a number of catabolic processes, which keep the cellular homeostasis by removing damaged and dysfunctional intracellular components. Impairment or misbalance of autophagy can lead to various diseases, such as neurodegeneration, infection diseases, and cancer. A central axis of autophagy is formed along the interactions of autophagy modifiers (Atg8-family proteins) with a variety of their cellular counter partners. Besides autophagy, Atg8-proteins participate in many other pathways, among which membrane trafficking and neuronal signaling are the most known. Despite the fact that autophagy modifiers are well-studied, as the small globular proteins show similarity to ubiquitin on a structural level, the mechanism of their interactions are still not completely understood. A thorough analysis and classification of all known mechanisms of Atg8-protein interactions could shed light on their functioning and connect the pathways involving Atg8-proteins. In this review, we present our views of the key features of the Atg8-proteins and describe the basic principles of their recognition and binding by interaction partners. We discuss affinity and selectivity of their interactions as well as provide perspectives for discovery of new Atg8-interacting proteins and therapeutic approaches to tackle major human diseases.
    Keywords:  Atg8; GABARAP; LC3; LIR motif; SAR; UBL; autophagy
    DOI:  https://doi.org/10.3390/cells9092008
  41. Behav Genet. 2020 Sep 05.
    Genetic Inactivation of Two-Pore Channel 1 Impairs Spatial Learning and Memory.
    Robert Theodor Mallmann, Norbert Klugbauer.
      Two-pore channels (TPCs) constitute a small family of cation channels that are localized in membranes of endosomal and lysosomal compartments. Although their roles for vesicular fusion and endolysosomal trafficking have been investigated, our knowledge on their expression pattern and higher order functions in the murine brain is still limited. Western blot analysis indicated a broad expression of TPC1 in the neocortex, cerebellum and hippocampus. In order to investigate the consequences of the genetic inactivation of TPC1, we performed a set of behavioural studies with TPC1-/- mice. TPC1-/- mice were analysed for an altered motor coordination and grip-strength, exploratory drive and anxiety as well as learning and memory. TPC1-/- mice did not show any differences in their exploratory drive or in their anxiety levels. There were also no differences in spontaneous activity or motor performance. However, the Morris water maze test uncovered a deficit in spatial learning and memory in TPC1-/- mice.
    Keywords:  Morris water maze; NAADP; Spatial learning; TPC1; Two-pore channel
    DOI:  https://doi.org/10.1007/s10519-020-10011-1
  42. J Cell Mol Med. 2020 Aug 30.
    The neurofibromatosis type I gene promotes autophagy via mTORC1 signalling pathway to enhance new bone formation after fracture.
    Qian Tan, Jiang-Yan Wu, Yao-Xi Liu, Kun Liu, Jin Tang, Wei-Hua Ye, Guang-Hui Zhu, Hai-Bo Mei, Ge Yang.
      Bone fracture is one of the most common injuries. Despite the high regenerative capacity of bones, failure of healing still occurs to near 10% of the patients. Herein, we aim to investigate the modulatory role of neurofibromatosis type I gene (NF1) to osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and new bone formation after fracture in a rat model. We studied the NF1 gene expression in normal and non-union bone fracture models. Then, we evaluated how NF1 overexpression modulated osteogenic differentiation of BMSCs, autophagy activity, mTORC1 signalling and osteoclastic bone resorption by qRT-PCR, Western blot and immunostaining assays. Finally, we injected lentivirus-NF1 (Lv-NF1) to rat non-union bone fracture model and analysed the bone formation process. The NF1 gene expression was significantly down-regulated in non-union bone fracture group, indicating NF1 is critical in bone healing process. In the NF1 overexpressing BMSCs, autophagy activity and osteogenic differentiation were significantly enhanced. Meanwhile, the NF1 overexpression inhibited mTORC1 signalling and osteoclastic bone resorption. In rat non-union bone fracture model, the NF1 overexpression significantly promoted bone formation during fracture healing. In summary, we proved the NF1 gene is critical in non-union bone healing, and NF1 overexpression promoted new bone formation after fracture by enhancing autophagy and inhibiting mTORC1 signalling. Our results may provide a novel therapeutic clue of promoting bone fracture healing.
    Keywords:  autophagy; fracture; osteogenesis; the neurofibromatosis type I gene
    DOI:  https://doi.org/10.1111/jcmm.15767
  43. Int J Mol Sci. 2020 Sep 01. pii: E6355. [Epub ahead of print]21(17):
    Assessing Lysosomal Disorders in the NGS Era: Identification of Novel Rare Variants.
    Marisa Encarnação, Maria Francisca Coutinho, Lisbeth Silva, Diogo Ribeiro, Souad Ouesleti, Teresa Campos, Helena Santos, Esmeralda Martins, Maria Teresa Cardoso, Laura Vilarinho, Sandra Alves.
      Lysosomal storage diseases (LSDs) are a heterogeneous group of genetic disorders with variable degrees of severity and a broad phenotypic spectrum, which may overlap with a number of other conditions. While individually rare, as a group LSDs affect a significant number of patients, placing an important burden on affected individuals and their families but also on national health care systems worldwide. Here, we present our results on the use of an in-house customized next-generation sequencing (NGS) panel of genes related to lysosome function as a first-line molecular test for the diagnosis of LSDs. Ultimately, our goal is to provide a fast and effective tool to screen for virtually all LSDs in a single run, thus contributing to decrease the diagnostic odyssey, accelerating the time to diagnosis. Our study enrolled a group of 23 patients with variable degrees of clinical and/or biochemical suspicion of LSD. Briefly, NGS analysis data workflow, followed by segregation analysis allowed the characterization of approximately 41% of the analyzed patients and the identification of 10 different pathogenic variants, underlying nine LSDs. Importantly, four of those variants were novel, and, when applicable, their effect over protein structure was evaluated through in silico analysis. One of the novel pathogenic variants was identified in the GM2A gene, which is associated with an ultra-rare (or misdiagnosed) LSD, the AB variant of GM2 Gangliosidosis. Overall, this case series highlights not only the major advantages of NGS-based diagnostic approaches but also, to some extent, its limitations ultimately promoting a reflection on the role of targeted panels as a primary tool for the prompt characterization of LSD patients.
    Keywords:  CLN7; GM2 Gangliosidosis; GM2A gene; bioinformatics analysis; diagnostics odyssey; lysosomal storage diseases (LSDs); molecular genetic testing (MGT); next-generation sequencing (NGS)
    DOI:  https://doi.org/10.3390/ijms21176355
  44. Dev Cell. 2020 Sep 01. pii: S1534-5807(20)30599-2. [Epub ahead of print]
    An Excitable Ras/PI3K/ERK Signaling Network Controls Migration and Oncogenic Transformation in Epithelial Cells.
    Huiwang Zhan, Sayak Bhattacharya, Huaqing Cai, Pablo A Iglesias, Chuan-Hsiang Huang, Peter N Devreotes.
      The Ras/PI3K/extracellular signal-regulated kinases (ERK) signaling network plays fundamental roles in cell growth, survival, and migration and is frequently activated in cancer. Here, we show that the activities of the signaling network propagate as coordinated waves, biased by growth factor, which drive actin-based protrusions in human epithelial cells. The network exhibits hallmarks of biochemical excitability: the annihilation of oppositely directed waves, all-or-none responsiveness, and refractoriness. Abrupt perturbations to Ras, PI(4,5)P2, PI(3,4)P2, ERK, and TORC2 alter the threshold, observations that define positive and negative feedback loops within the network. Oncogenic transformation dramatically increases the wave activity, the frequency of ERK pulses, and the sensitivity to EGF stimuli. Wave activity was progressively enhanced across a series of increasingly metastatic breast cancer cell lines. The view that oncogenic transformation is a shift to a lower threshold of excitable Ras/PI3K/ERK network, caused by various combinations of genetic insults, can facilitate the assessment of cancer severity and effectiveness of interventions.
    Keywords:  ERK; PI(3,4)P2; PI(4,5)P2; PI3K; Ras; excitability; oncogenic transformation; threshold; wave
    DOI:  https://doi.org/10.1016/j.devcel.2020.08.001
  45. Nature. 2020 Sep 02.
    In-cell architecture of the nuclear pore and snapshots of its turnover.
    Matteo Allegretti, Christian E Zimmerli, Vasileios Rantos, Florian Wilfling, Paolo Ronchi, Herman K H Fung, Chia-Wei Lee, Wim Hagen, Beata Turoňová, Kai Karius, Mandy Börmel, Xiaojie Zhang, Christoph W Müller, Yannick Schwab, Julia Mahamid, Boris Pfander, Jan Kosinski, Martin Beck.
      Nuclear pore complexes (NPCs) fuse the inner and outer membranes of the nuclear envelope. They comprise hundreds of nucleoporins (Nups) that assemble into multiple subcomplexes and form large central channels for nucleocytoplasmic exchange1,2. How this architecture facilitates messenger RNA export, NPC biogenesis and turnover remains poorly understood. Here we combine in situ structural biology and integrative modelling with correlative light and electron microscopy and molecular perturbation to structurally analyse NPCs in intact Saccharomyces cerevisiae cells within the context of nuclear envelope remodelling. We find an in situ conformation and configuration of the Nup subcomplexes that was unexpected from the results of previous in vitro analyses. The configuration of the Nup159 complex appears critical to spatially accommodate its function as an mRNA export platform, and as a mediator of NPC turnover. The omega-shaped nuclear envelope herniae that accumulate in nup116Δ cells3 conceal partially assembled NPCs lacking multiple subcomplexes, including the Nup159 complex. Under conditions of starvation, herniae of a second type are formed that cytoplasmically expose NPCs. These results point to a model of NPC turnover in which NPC-containing vesicles bud off from the nuclear envelope before degradation by the autophagy machinery. Our study emphasizes the importance of investigating the structure-function relationship of macromolecular complexes in their cellular context.
    DOI:  https://doi.org/10.1038/s41586-020-2670-5
  46. Dev Biol. 2020 Aug 28. pii: S0012-1606(20)30237-2. [Epub ahead of print]
    mTOR plays a pivotal role in multiple processes of enamel organ development principally through the mTORC1 pathway and in part via regulating cytoskeleton dynamics.
    Xuguang Nie, Jinxuan Zheng, Michael Cruciger, Peixin Yang, Jeremy J Mao.
      We herein report that deletion of mTOR in dental epithelia caused defective development of multiple cell layers of the enamel organ, which culminated in tooth malformation and cystogenesis. Specifically, cells of the stellate reticulum and stratum intermedium were poorly formed, resulting in cystic change. The pre-ameloblasts failed to elongate along the apical-basal axis and persisted vigorous expression of Sox2 and P63, which are normally downregulated during cytodifferentiation. Expression of amelogenic markers was also attenuated in mutants. Cell proliferation and cell sizes in mutants were significantly reduced over time. Importantly, we found reduced amounts and aberrant aggregations of cytoskeletal components in mutants, along with attenuated expression of cytoskeleton regulator Cdc42, whose epithelial deletion causes a similar phenotype. Moreover, disruption of actin assembly in an organ culture system affected cell proliferation and cytodifferentiation of tooth germs, supporting a causative role of mTOR-regulated cytoskeleton dynamics for the observed phenotype of mTOR mutant mice. In further support of this view, we showed that mTOR overactivation caused increased cytoskeletal component synthesis and assembly, along with accelerated cytodifferentiation in the enamel organ. Finally, we demonstrated that mTOR regulated enamel organ development principally through the mTORC1 pathway.
    Keywords:  Development; Enamel organ; Mouse; Tsc1; mTOR
    DOI:  https://doi.org/10.1016/j.ydbio.2020.08.010
  47. Eur J Med Genet. 2020 Sep 01. pii: S1769-7212(20)30492-4. [Epub ahead of print] 104060
    TSC1 intragenic deletion transmitted from a mosaic father to two siblings with cardiac rhabdomyomas: Identification of two aberrant transcripts.
    Hiroki Uchiyama, Yohei Masunaga, Takamichi Ishikawa, Tetsuya Fukuoka, Maki Fukami, Hirotomo Saitsu, Tsutomu Ogat.
      Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder characterized by non-cancerous tumors in multiple organs including the brain, kidney, lung, heart, and skin. We encountered a Japanese family consisting of two siblings (a four-year-old boy and a one-year-old girl) with multiple cardiac rhabdomyomas conveying a high risk of TSC and apparently unaffected sibling (a two-year-old girl) and parents. Whole exome sequencing and application of Integrative Genomic Viewer revealed an identical intragenic TSC1 deletion with the breakpoints on intron 15 and exon 19 in the affected siblings, but not in the apparently unaffected sibling and parents. Subsequently, PCR-based analyses were performed using primers flanking the deletion, showing that the deletion was also present in the father and that the deletion occurred between chr9:135,777,038 (bp) and chr9:135,780,540 (bp) in association with a one bp overlap. Furthermore, RT-PCR analyses were carried out using lymphoblastoid cell lines, revealing a major in-frame insertion/deletion transcript produced by aberrant splicing using a cryptic ″ag″ splice acceptor motif at intron 15 (r.1998_2438delinsTTCATTAGGTGG) and a minor frameshift transcript generated by aberrant splicing between exon 15 and exon 20 (r.1998_2502del, p.Lys666Asnfs*15) in the affected siblings. These findings imply that the intragenic deletion producing two aberrant transcripts was generated as a somatic pathogenic variant involving the germline in the father and was transmitted to the affected siblings.
    Keywords:  Aberrant transcript; Cardiac rhabdomyoma; Germline mosaicism; Intragenic deletion; TSC1, tuberous sclerosis complex
    DOI:  https://doi.org/10.1016/j.ejmg.2020.104060
  48. Clin Genet. 2020 Aug 19.
    ASAH1-related disorders: Description of 15 novel pediatric patients and expansion of the clinical phenotype.
    Iman G Mahmoud, Mohamed A Elmonem, Maha S Zaki, Areef Ramadan, Nihal M Al-Menabawy, Aya El-Gamal, Lobna Mansour, Mahmoud Y Issa, Mohamed S Abdel-Hamid, Sawsan Abdel-Hady, Iman Khalifa, Ahmed Ibrahim, Alexander Solyom, Arndt Rolfs, Laila Selim.
      Acid ceramidase deficiency is an orphan lysosomal disorder caused by ASAH1 pathogenic variants and presenting with either Farber disease or spinal muscle atrophy with progressive myoclonic epilepsy (SMA-PME). Phenotypic and genotypic features are rarely explored beyond the scope of case reports. Furthermore, the new biomarker C26-Ceramide requires validation in a clinical setting. We evaluated the clinical, biomarker and genetic spectrum of 15 Egyptian children from 14 unrelated families with biallelic pathogenic variants in ASAH1 (12 Farber and 3 SMA-PME). Recruited children were nine females/six males ranging in age at diagnosis from 13 to 118 months. We detected ASAH1 pathogenic variants in all 30 alleles including three novel variants (c.1126A>G (p.Thr376Ala), c.1205G>A (p.Arg402Gln), exon-5-deletion). Both total C26-Ceramide and its trans- isomer showed 100% sensitivity for the detection of ASAH1-related disorders in tested patients. A 10-year-old girl with the novel variant c.1205G>A (p.Arg402Gln) presented with a new peculiar phenotype of PME without muscle atrophy. We expanded the phenotypic spectrum of ASAH1-related disorders and validated the biomarker C26-Ceramide for supporting diagnosis in symptomatic patients.
    Keywords:  ASAH1; C26-ceramide; Farber disease; SMA-PME; acid ceramidase
    DOI:  https://doi.org/10.1111/cge.13834
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