bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2022–01–16
43 papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing
  1. PP2A-dependent TFEB activation is blocked by PIKfyve-induced mTORC1 activity.
  2. A Journey towards Understanding the Molecular Pathology and Developing Therapies for Lysosomal Storage Disorders.
  3. Drosophila D-idua Reduction Mimics Mucopolysaccharidosis Type I Disease-Related Phenotypes.
  4. Tributyltin inhibits autophagy by decreasing lysosomal acidity in SH-SY5Y cells.
  5. Emerging Lysosomal Functions for Photoreceptor Cell Homeostasis and Survival.
  6. Lysosomal potassium channels.
  7. EBV LMP1-activated mTORC1 and mTORC2 Coordinately Promote Nasopharyngeal Cancer Stem Cell Properties.
  8. Targeting mTOR Signaling in Type 2 Diabetes Mellitus and Diabetes Complications.
  9. Elevated granulocyte-colony stimulating factor and hematopoietic stem cell mobilization in Niemann-Pick Type C1 disease.
  10. GATOR2 complex-mediated amino acid signaling regulates brain myelination.
  11. Detection and quantification of the vacuolar H+ATPase using the Legionella effector protein SidK.
  12. Abnormal activation of yap/Taz contributes to the pathogenesis of tuberous sclerosis complex.
  13. Bioengineered Cystinotic Kidney Tubules Recapitulate a Nephropathic Phenotype.
  14. Impaired Retrograde Transport Due to Lack of TBC1D5 Contributes to the Trafficking Defect of Lysosomal Cathepsins in Ischemic/Hypoxic Cardiomyocytes.
  15. Loss of TREM2 rescues hyperactivation of microglia, but not lysosomal deficits and neurotoxicity in models of progranulin deficiency.
  16. AAV9/MFSD8 gene therapy is effective in preclinical models of neuronal ceroid lipofuscinosis type 7 disease.
  17. RABEP1/Rabaptin5: a link between autophagy and early endosome homeostasis.
  18. Annexin A6 and NPC1 regulate LDL-inducible cell migration and distribution of focal adhesions.
  19. JIP3 links lysosome transport to regulation of multiple components of the axonal cytoskeleton.
  20. Inhibition of Autophagy via Lysosomal Impairment Enhances Cytotoxicity of Fullerenol under Starvation Condition.
  21. Mosaic dysfunction of mitophagy in mitochondrial muscle disease.
  22. Chronic lithium administration in a mouse model for Krabbe disease.
  23. Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins.
  24. Brain and visceral gene editing of mucopolysaccharidosis I mice by nasal delivery of the CRISPR/Cas9 system.
  25. CDC42 controlled apical-basal polarity regulates intestinal stem cell to transit amplifying cell fate transition via YAP-EGF-mTOR signaling.
  26. RNA binding protein HuD promotes autophagy and tumor stress survival by suppressing mTORC1 activity and augmenting ARL6IP1 levels.
  27. Substrate-induced differential degradation and partitioning of the two tryptophan permeases Tat1 and Tat2 into eisosomes in Saccharomyces cerevisiae.
  28. Translational and post-translational control of human naïve versus primed pluripotency.
  29. In Cellulo Crystallization of Human Neuraminidase 1 and Biological Roles of N-Glycans.
  30. Methylmercury induces lysosomal membrane permeabilization through JNK-activated Bax lysosomal translocation in neuronal cells.
  31. Coordination of inter-organelle communication and lipid fluxes by OSBP-related proteins.
  32. Structural advances in sterol-sensing domain-containing proteins.
  33. Tsc2 mutation induces renal tubular cell nonautonomous disease.
  34. Selectivity of mTOR-Phosphatidic Acid Interactions Is Driven by Acyl Chain Structure and Cholesterol.
  35. In Vitro and In Vivo Models to Study Nephropathic Cystinosis.
  36. Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice.
  37. Plant virus infection disrupts vacuolar acidification and autophagic degradation for the effective infection.
  38. SERPINB3 (SCCA1) inhibits cathepsin L and lysoptosis, protecting cervical cancer cells from chemoradiation.
  39. Site-specific ubiquitination of MLKL targets it to endosomes and targets Listeria and Yersinia to the lysosomes.
  40. Disruption of PIKFYVE causes congenital cataract in human and zebrafish.
  41. Prion-like α-synuclein pathology in the brain of infants with Krabbe disease.
  42. Down-regulation of the brain-specific cell-adhesion molecule contactin-3 in tuberous sclerosis complex during the early postnatal period.
  43. Interactome analysis of human phospholipase D and phosphatidic acid-associated protein network.
  1. Mol Biol Cell. 2022 Jan 12. mbcE21060309
    PP2A-dependent TFEB activation is blocked by PIKfyve-induced mTORC1 activity.
    Junya Hasegawa, Emi Tokuda, Yao Yao, Takehiko Sasaki, Ken Inoki, Lois S Weisman.
      Transcriptional factor EB (TFEB) is a master regulator of genes required for autophagy and lysosomal function. The nuclear localization of TFEB is blocked by the mechanistic target of rapamycin complex 1 (mTORC1)-dependent phosphorylation of TFEB at multiple sites including Ser-211. Here we show that inhibition of PIKfyve, which produces phosphatidylinositol 3,5-bisphosphate on endosomes and lysosomes, causes a loss of Ser-211 phosphorylation and concomitant nuclear localization of TFEB. We found that while mTORC1 activity toward S6K1, as well as other major mTORC1 substrates, is not impaired, PIKfyve inhibition specifically impedes the interaction of TFEB with mTORC1. This suggests that mTORC1 activity on TFEB is selectively inhibited due to loss of mTORC1 access to TFEB. In addition, we found that TFEB activation during inhibition of PIKfyve relies on the ability of protein phosphatase 2A (PP2A) but not calcineurin/PPP3, to dephosphorylate TFEB Ser-211. Thus, when PIKfyve is inhibited, PP2A is dominant over mTORC1 for control of TFEB phosphorylation at Ser-S211. Together these findings suggest that mTORC1 and PP2A have opposing roles on TFEB via phosphorylation and dephosphorylation of Ser-211, respectively, and further, that PIKfyve inhibits TFEB activity by facilitating mTORC1-dependent phosphorylation of TFEB.
    DOI:  https://doi.org/10.1091/mbc.E21-06-0309
  2. Cells. 2021 Dec 23. pii: 36. [Epub ahead of print]11(1):
    A Journey towards Understanding the Molecular Pathology and Developing Therapies for Lysosomal Storage Disorders.
    Ritva Tikkanen.
      Lysosomal storage disorders (LSDs) are rare, monogenic diseases characterized by aberrant lysosomes with storage material [...].
    DOI:  https://doi.org/10.3390/cells11010036
  3. Cells. 2021 Dec 31. pii: 129. [Epub ahead of print]11(1):
    Drosophila D-idua Reduction Mimics Mucopolysaccharidosis Type I Disease-Related Phenotypes.
    Concetta De Filippis, Barbara Napoli, Laura Rigon, Giulia Guarato, Reinhard Bauer, Rosella Tomanin, Genny Orso.
      Deficit of the IDUA (α-L-iduronidase) enzyme causes the lysosomal storage disorder mucopolysaccharidosis type I (MPS I), a rare pediatric neurometabolic disease, due to pathological variants in the IDUA gene and is characterized by the accumulation of the undegraded mucopolysaccharides heparan sulfate and dermatan sulfate into lysosomes, with secondary cellular consequences that are still mostly unclarified. Here, we report a new fruit fly RNAi-mediated knockdown model of a IDUA homolog (D-idua) displaying a phenotype mimicking some typical molecular features of Lysosomal Storage Disorders (LSD). In this study, we showed that D-idua is a vital gene in Drosophila and that ubiquitous reduction of its expression leads to lethality during the pupal stage, when the precise degradation/synthesis of macromolecules, together with a functional autophagic pathway, are indispensable for the correct development to the adult stage. Tissue-specific analysis of the D-idua model showed an increase in the number and size of lysosomes in the brain and muscle. Moreover, the incorrect acidification of lysosomes led to dysfunctional lysosome-autophagosome fusion and the consequent block of autophagy flux. A concomitant metabolic drift of glycolysis and lipogenesis pathways was observed. After starvation, D-idua larvae showed a quite complete rescue of both autophagy/lysosome phenotypes and metabolic alterations. Metabolism and autophagy are strictly interconnected vital processes that contribute to maintain homeostatic control of energy balance, and little is known about this regulation in LSDs. Our results provide new starting points for future investigations on the disease's pathogenic mechanisms and possible pharmacological manipulations.
    Keywords:  D-idua; Drosophila melanogaster; Hurler syndrome; IDUA; MPS I; RNAi; fly models; lysosomal storage disorders; mucopolysaccharidosis type I
    DOI:  https://doi.org/10.3390/cells11010129
  4. Biochem Biophys Res Commun. 2022 Jan 04. pii: S0006-291X(21)01769-1. [Epub ahead of print]592 31-37
    Tributyltin inhibits autophagy by decreasing lysosomal acidity in SH-SY5Y cells.
    Shunichi Hatamiya, Masatsugu Miyara, Yaichiro Kotake.
      Tributyltin (TBT) is an environmental pollutant that remains in marine sediments and is toxic to mammals. For example, TBT elicits neurotoxic and immunosuppressive effects on rats. However, it is not entirely understood how TBT causes toxicity. Autophagy plays a pivotal role in protein quality control and eliminates aggregated proteins and damaged organelles. We previously reported that TBT dephosphorylates mammalian target of rapamycin (mTOR), which may be involved in enhancement of autophagosome synthesis, in primary cultures of cortical neurons. Autophagosomes can accumulate due to enhancement of autophagosome synthesis or inhibition of autophagic degradation, and we did not clarify whether TBT alters autophagic flux. Here, we investigated the mechanism by which TBT causes accumulation of autophagosomes in SH-SY5Y cells. TBT inhibited autophagy without affecting autophagosome-lysosome fusion before it caused cell death. TBT dramatically decreased the acidity of lysosomes without affecting lysosomal membrane integrity. TBT decreased the mature protein level of cathepsin B, and this may be related to the decrease in lysosomal acidity. These results suggest that TBT inhibits autophagic degradation by decreasing lysosomal acidity. Autophagy impairment may be involved in the mechanism underlying neuronal death and/or T-cell-dependent thymus atrophy induced by TBT.
    Keywords:  Autophagy; Lysosome; Tributyltin
    DOI:  https://doi.org/10.1016/j.bbrc.2021.12.118
  5. Cells. 2021 Dec 26. pii: 60. [Epub ahead of print]11(1):
    Emerging Lysosomal Functions for Photoreceptor Cell Homeostasis and Survival.
    Manuela Santo, Ivan Conte.
      Lysosomes are membrane-bound cell organelles that respond to nutrient changes and are implicated in cell homeostasis and clearance mechanisms, allowing effective adaptation to specific cellular needs. The relevance of the lysosome has been elucidated in a number of different contexts. Of these, the retina represents an interesting scenario to appreciate the various functions of this organelle in both physiological and pathological conditions. Growing evidence suggests a role for lysosome-related mechanisms in retinal degeneration. Abnormal lysosomal activation or inhibition has dramatic consequences on photoreceptor cell homeostasis and impacts extensive cellular function, which in turn affects vision. Based on these findings, a series of therapeutic methods targeting lysosomal processes could offer treatment for blindness conditions. Here, we review the recent findings on membrane trafficking, subcellular organization, mechanisms by which lysosome/autophagy pathway impairment affects photoreceptor cell homeostasis and the recent advances on developing efficient lysosomal-based therapies for retinal disorders.
    Keywords:  autophagy; lysosome; membrane trafficking; photoreceptors; retinal degeneration
    DOI:  https://doi.org/10.3390/cells11010060
  6. Cell Calcium. 2022 Jan 06. pii: S0143-4160(22)00011-2. [Epub ahead of print]102 102536
    Lysosomal potassium channels.
    Yi Wu, Mengnan Xu, Pingping Wang, Alia Kazim Rizvi Syeda, Peng Huang, Xian-Ping Dong.
      The lysosome is an important membrane-bound acidic organelle that is regarded as the degradative center as well as multifunctional signaling hub. It digests unwanted macromolecules, damaged organelles, microbes, and other materials derived from endocytosis, autophagy, and phagocytosis. To function properly, the ionic homeostasis and membrane potential of the lysosome are strictly regulated by transporters and ion channels. As the most abundant cation inside the cell, potassium ions (K+) are vital for lysosomal membrane potential and lysosomal calcium (Ca2+) signaling. However, our understanding about how lysosomal K+homeostasis is regulated and what are the functions of K+in the lysosome is very limited. Currently, two lysosomal K+channels have been identified: large-conductance Ca2+-activated K+channel (BK) and transmembrane Protein 175 (TMEM175). In this review, we summarize recent development in our understanding of K+ homeostasis and K+channels in the lysosome. We hope to guide the readers into a more in-depth discussion of lysosomal K+ channels in lysosomal physiology and human diseases.
    Keywords:  BK channel; Lysosome; TMEM175; TRPML1
    DOI:  https://doi.org/10.1016/j.ceca.2022.102536
  7. J Virol. 2022 Jan 12. jvi0194121
    EBV LMP1-activated mTORC1 and mTORC2 Coordinately Promote Nasopharyngeal Cancer Stem Cell Properties.
    Nannan Zhu, Qian Wang, Zhidong Wu, Yan Wang, Mu-Sheng Zeng, Yan Yuan.
      Epstein-Barr Virus (EBV) is associated with several malignant diseases, including Burkitt's lymphoma, nasopharyngeal carcinoma (NPC), certain types of lymphomas, and a portion of gastric cancers. Virus-encoded oncoprotein LMP1 induces the epithelial-to-mesenchymal transition (EMT), leading to cancer stem cell formation. In the current study, we investigated how LMP1 contributes to cancer stem cell development in NPC. We found that LMP1 plays an essential role in acquiring CSC characteristics, including tumor initiation, metastasis, and therapeutic resistance by activating the PI3K/mTOR/Akt signaling pathway. We dissected the functions of distinct signaling (mTORC1 and mTORC2) in the acquisition of different CSC characteristics. Side population (SP) formation, which represents the chemotherapy resistance feature of CSC, requires mTORC1 signaling. Tumor initiation capability is mainly attributed to mTORC2, which confers on NPC the capabilities of proliferation and survival by activating mTORC2 downstream genes c-Myc. Both mTORC1 and mTORC2 enhance cell migration and invasion of NPC cells, suggesting that mTORC1/2 co-regulate metastasis of NPC. The revelation of the roles of the mTOR signaling pathways in distinct tumorigenic features provides a guideline for designing efficient therapies by choosing specific mTOR inhibitors targeting mTORC1, mTORC2, or both to achieve durable remission of NPC in patients. Importance LMP1 endows NPC to gain cancer stem cell characteristics through activating mTORC1 and mTORC2 pathways. The different mTOR pathways are responsible for distinct tumorigenic features. Rapamycin-insensitive mTORC1 is essential for CSC drug resistance. NPC tumor initiation capacity is mainly attributed to mTORC2 signaling. mTORC1 and mTORC2 co-regulate NPC cell migration and invasion. The revelation of the roles of mTOR signaling in NPC CSC establishment has implications for novel therapeutic strategies to treat relapsed and metastatic NPC and achieve durable remission.
    DOI:  https://doi.org/10.1128/jvi.01941-21
  8. Curr Drug Targets. 2022 Jan 11.
    Targeting mTOR Signaling in Type 2 Diabetes Mellitus and Diabetes Complications.
    Lin Yang, Zhixin Zhang, Doudou Wang, Yu Jiang, Ying Liu.
      The mechanistic target of rapamycin (mTOR) is a pivotal regulator of cell metabolism and growth. In the form of two different multi-protein complexes, mTORC1 and mTORC2, mTOR integrates cellular energy, nutrient and hormonal signals to regulate cellular metabolic homeostasis. In type 2 diabetes mellitus (T2DM) aberrant mTOR signaling underlies its pathological conditions and end-organ complications. Substantial evidence suggests that two mTOR-mediated signaling schemes, mTORC1-p70S6 kinase 1 (S6K1) and mTORC2-protein kinase B (AKT), play a critical role in insulin sensitivity and that their dysfunction contributes to development of T2DM. This review summaries our current understanding of the role of mTOR signaling in T2DM and its associated complications, as well as the potential use of mTOR inhibitors in treatment of T2DM.
    Keywords:  diabetes complications; mTOR inhibitor; mTORC1; mTORC2; type 2 diabetes mellitus
    DOI:  https://doi.org/10.2174/1389450123666220111115528
  9. J Lipid Res. 2022 Jan 07. pii: S0022-2275(21)00150-4. [Epub ahead of print] 100167
    Elevated granulocyte-colony stimulating factor and hematopoietic stem cell mobilization in Niemann-Pick Type C1 disease.
    Anouk G Groenen, Anouk M La Rose, Mengying Li, Venetia Bazioti, Arthur F Svendsen, Niels J Kloosterhuis, Albertina Ausema, Alle Pranger, M Rebecca Heiner-Fokkema, Klary E Niezen-Koning, Tom Houben, Ronit Shiri-Sverdlov, Marit Westerterp.
      Niemann-Pick Type C1 (NPC1) disease is a progressive lysosomal storage disorder caused by mutations of the NPC1 gene. While neurodegeneration is the most severe symptom, a large proportion of NPC1 patients also present with splenomegaly, which has been attributed to cholesterol and glycosphingolipid accumulation in late endosomes and lysosomes. However, recent data also reveal an increase in the inflammatory monocyte subset in the Npc1nih mouse model expressing a Npc1-null allele. We evaluated the contribution of hematopoietic cells to splenomegaly in NPC1 disease under conditions of hypercholesterolemia. We transplanted Npc1nih (Npc1 null-mutation) or Npc1wt bone marrow into Ldlr-/- mice and fed these mice a cholesterol-rich Western-type diet (WTD). At 9 weeks after bone marrow transplant (BMT), on a chow diet, the Npc1 null-mutation increased plasma granulocyte-colony stimulating factor (G-CSF) by twofold and caused mild neutrophilia. At 18 weeks after BMT, including 9 weeks of WTD feeding, the Npc1 mutation increased G-csf mRNA levels by ∼5-fold in splenic monocytes/macrophages accompanied by a ∼4-fold increase in splenic neutrophils compared to controls. We also observed ∼5-fold increased long-term and short-term hematopoietic stem cells (HSCs) in the spleen, and a ∼30-75% decrease of these populations in BM, reflecting HSC mobilization, presumably downstream of elevated G-CSF. In line with these data, four patients with NPC1 disease showed higher plasma G-CSF compared to age- and gender-matched healthy controls. In conclusion, we show elevated G-CSF levels and HSC mobilization in the setting of an Npc1-null mutation, and propose that this contributes to splenomegaly in patients with NPC1 disease.
    Keywords:  Animal models; Cholesterol/Trafficking; Macrophages/Monocytes; Neutrophils; Storage diseases; hematopoietic stem cells; inflammation; splenomegaly
    DOI:  https://doi.org/10.1016/j.jlr.2021.100167
  10. Proc Natl Acad Sci U S A. 2022 Jan 18. pii: e2110917119. [Epub ahead of print]119(3):
    GATOR2 complex-mediated amino acid signaling regulates brain myelination.
    Zongyan Yu, Zhiwen Yang, Guoru Ren, Yingjie Wang, Xiang Luo, Feiyan Zhu, Shouyang Yu, Lanlan Jia, Mina Chen, Paul F Worley, Bo Xiao.
      Amino acids are essential for cell growth and metabolism. Amino acid and growth factor signaling pathways coordinately regulate the mechanistic target of rapamycin complex 1 (mTORC1) kinase in cell growth and organ development. While major components of amino acid signaling mechanisms have been identified, their biological functions in organ development are unclear. We aimed to understand the functions of the critically positioned amino acid signaling complex GAP activity towards Rags 2 (GATOR2) in brain development. GATOR2 mediates amino acid signaling to mTORC1 by directly linking the amino acid sensors for arginine and leucine to downstream signaling complexes. Now, we report a role of GATOR2 in oligodendrocyte myelination in postnatal brain development. We show that the disruption of GATOR2 complex by genetic deletion of meiosis regulator for oocyte development (Mios, encoding a component of GATOR2) selectively impairs the formation of myelinating oligodendrocytes, thus brain myelination, without apparent effects on the formation of neurons and astrocytes. The loss of Mios impairs cell cycle progression of oligodendrocyte precursor cells, leading to their reduced proliferation and differentiation. Mios deletion manifests a cell type-dependent effect on mTORC1 in the brain, with oligodendroglial mTORC1 selectively affected. However, the role of Mios/GATOR2 in oligodendrocyte formation and myelination involves mTORC1-independent function. This study suggests that GATOR2 coordinates amino acid and growth factor signaling to regulate oligodendrocyte myelination.
    Keywords:  GATOR2; Mios; amino acid signaling; myelination; oligodendrocytes
    DOI:  https://doi.org/10.1073/pnas.2110917119
  11. J Cell Biol. 2022 Mar 07. pii: e202107174. [Epub ahead of print]221(3):
    Detection and quantification of the vacuolar H+ATPase using the Legionella effector protein SidK.
    Michelle E Maxson, Yazan M Abbas, Jing Ze Wu, Jonathan D Plumb, Sergio Grinstein, John L Rubinstein.
      Acidification of secretory and endocytic organelles is required for proper receptor recycling, membrane traffic, protein degradation, and solute transport. Proton-pumping vacuolar H+ ATPases (V-ATPases) are responsible for this luminal acidification, which increases progressively as secretory and endocytic vesicles mature. An increasing density of V-ATPase complexes is thought to account for the gradual decrease in pH, but available reagents have not been sufficiently sensitive or specific to test this hypothesis. We introduce a new probe to localize and quantify V-ATPases. The probe is derived from SidK, a Legionella pneumophila effector protein that binds to the V-ATPase A subunit. We generated plasmids encoding fluorescent chimeras of SidK1-278, and labeled recombinant SidK1-278 with Alexa Fluor 568 to visualize and quantify V-ATPases with high specificity in live and fixed cells, respectively. We show that V-ATPases are acquired progressively during phagosome maturation, that they distribute in discrete membrane subdomains, and that their density in lysosomes depends on their subcellular localization.
    DOI:  https://doi.org/10.1083/jcb.202107174
  12. Hum Mol Genet. 2022 Jan 06. pii: ddab374. [Epub ahead of print]
    Abnormal activation of yap/Taz contributes to the pathogenesis of tuberous sclerosis complex.
    Bethany K Terry, Raehee Park, Seo-Hee Cho, Peter B Crino, Seonhee Kim.
      The multi-systemic genetic disorder tuberous sclerosis complex (TSC) impacts multiple neurodevelopmental processes including neuronal morphogenesis, neuronal migration, myelination, and gliogenesis. These alterations contribute to the development of cerebral cortex abnormalities and malformations. Although TSC is caused by mTORC1 hyperactivation, cognitive and behavioral impairments are not improved through mTORC1 targeting, making the study of the downstream effectors of this complex important for understanding the mechanisms underlying TSC. As mTORC1 has been shown to promote the activity of the transcriptional co-activator Yap, we hypothesized that altered Yap/Taz signaling contributes to the pathogenesis of TSC. We first observed that the level of Yap/Taz are increased in a human cortical tuber sample and in embryonic cortices of Tsc2 conditional knockout (cKO) mice. Next, to determine how abnormal upregulation of Yap/Taz impacts the neuropathology of TSC, we deleted Yap/Taz in Tsc2 cKO mice. Importantly, Yap/Taz/Tsc2 tcKO animals show reduced cortical thickness and cortical neuron cell size, despite the persistence of high mTORC1 activity, suggesting that Yap/Taz play a downstream role in cytomegaly. Furthermore, Yap/Taz/Tsc2 tcKO significantly restored cortical and hippocampal lamination defects and reduced hippocampal heterotopia formation. Finally, the loss of Yap/Taz increased the distribution of myelin basic protein in Tsc2 cKO animals, consistent with an improvement in myelination. Overall, our results indicate that targeting Yap/Taz lessens the severity of neuropathology in a TSC animal model. This study is the first to implicate Yap/Taz as contributors to cortical pathogenesis in TSC and therefore as potential novel targets in the treatment of this disorder.
    DOI:  https://doi.org/10.1093/hmg/ddab374
  13. Cells. 2022 Jan 05. pii: 177. [Epub ahead of print]11(1):
    Bioengineered Cystinotic Kidney Tubules Recapitulate a Nephropathic Phenotype.
    Elena Sendino Garví, Rosalinde Masereeuw, Manoe J Janssen.
      Nephropathic cystinosis is a rare and severe disease caused by disruptions in the CTNS gene. Cystinosis is characterized by lysosomal cystine accumulation, vesicle trafficking impairment, oxidative stress, and apoptosis. Additionally, cystinotic patients exhibit weakening and leakage of the proximal tubular segment of the nephrons, leading to renal Fanconi syndrome and kidney failure early in life. Current in vitro cystinotic models cannot recapitulate all clinical features of the disease which limits their translational value. Therefore, the development of novel, complex in vitro models that better mimic the disease and exhibit characteristics not compatible with 2-dimensional cell culture is of crucial importance for novel therapies development. In this study, we developed a 3-dimensional bioengineered model of nephropathic cystinosis by culturing conditionally immortalized proximal tubule epithelial cells (ciPTECs) on hollow fiber membranes (HFM). Cystinotic kidney tubules showed lysosomal cystine accumulation, increased autophagy and vesicle trafficking deterioration, the impairment of several metabolic pathways, and the disruption of the epithelial monolayer tightness as compared to control kidney tubules. In particular, the loss of monolayer organization and leakage could be mimicked with the use of the cystinotic kidney tubules, which has not been possible before, using the standard 2-dimensional cell culture. Overall, bioengineered cystinotic kidney tubules recapitulate better the nephropathic phenotype at a molecular, structural, and functional proximal tubule level compared to 2-dimensional cell cultures.
    Keywords:  3-dimensional models; autophagy; hollow fiber membrane; lysosomal storage disease; nephropathic cystinosis
    DOI:  https://doi.org/10.3390/cells11010177
  14. Front Cardiovasc Med. 2021 ;8 796254
    Impaired Retrograde Transport Due to Lack of TBC1D5 Contributes to the Trafficking Defect of Lysosomal Cathepsins in Ischemic/Hypoxic Cardiomyocytes.
    Lin Cui, Qiong Zhang, Yao Huang, Lei Yang, Junhui Zhang, Xupin Jiang, Jiezhi Jia, Yanling Lv, Dongxia Zhang, Yuesheng Huang.
      Lysosomal dysfunction has been found in many pathological conditions, and methods to improve lysosomal function have been reported to be protective against infarcted hearts. However, the mechanisms underlying lysosomal dysfunction caused by ischemic injury are far less well-established. The retromer complex is implicated in the trafficking of cation-independent mannose 6-phosphate receptor (CI-MPR), which is an important protein tag for the proper transport of lysosomal contents and therefore is important for the maintenance of lysosomal function. In this study, we found that the function of retrograde transport in cardiomyocytes was impaired with ischemia/hypoxia (I/H) treatment, which resulted in a decrease in CI-MPR and an abnormal distribution of lysosomal cathepsins. I/H treatment caused a reduction in TBC1D5 and a blockade of the Rab7 membrane cycle, which impeded retromer binding to microtubules and motor proteins, resulting in an impairment of retrograde transport and a decrease in CI-MPR. We also established that TBC1D5 was an important regulator of the distribution of lysosomal cathepsins. Our findings shed light on the regulatory role of retromer in ischemic injury and uncover the regulatory mechanism of TBC1D5 over retromer.
    Keywords:  CI-MPR; TBC1D5; cardiomyocyte; ischemia/hypoxia; lysosome; retromer
    DOI:  https://doi.org/10.3389/fcvm.2021.796254
  15. EMBO J. 2022 Jan 12. e109108
    Loss of TREM2 rescues hyperactivation of microglia, but not lysosomal deficits and neurotoxicity in models of progranulin deficiency.
    Anika Reifschneider, Sophie Robinson, Bettina van Lengerich, Johannes Gnörich, Todd Logan, Steffanie Heindl, Miriam A Vogt, Endy Weidinger, Lina Riedl, Karin Wind, Artem Zatcepin, Ida Pesämaa, Sophie Haberl, Brigitte Nuscher, Gernot Kleinberger, Julien Klimmt, Julia K Götzl, Arthur Liesz, Katharina Bürger, Matthias Brendel, Johannes Levin, Janine Diehl-Schmid, Jung Suh, Gilbert Di Paolo, Joseph W Lewcock, Kathryn M Monroe, Dominik Paquet, Anja Capell, Christian Haass.
      Haploinsufficiency of the progranulin (PGRN)-encoding gene (GRN) causes frontotemporal lobar degeneration (GRN-FTLD) and results in microglial hyperactivation, TREM2 activation, lysosomal dysfunction, and TDP-43 deposition. To understand the contribution of microglial hyperactivation to pathology, we used genetic and pharmacological approaches to suppress TREM2-dependent transition of microglia from a homeostatic to a disease-associated state. Trem2 deficiency in Grn KO mice reduced microglia hyperactivation. To explore antibody-mediated pharmacological modulation of TREM2-dependent microglial states, we identified antagonistic TREM2 antibodies. Treatment of macrophages from GRN-FTLD patients with these antibodies led to reduced TREM2 signaling due to its enhanced shedding. Furthermore, TREM2 antibody-treated PGRN-deficient microglia derived from human-induced pluripotent stem cells showed reduced microglial hyperactivation, TREM2 signaling, and phagocytic activity, but lysosomal dysfunction was not rescued. Similarly, lysosomal dysfunction, lipid dysregulation, and glucose hypometabolism of Grn KO mice were not rescued by TREM2 ablation. Synaptic loss and neurofilament light-chain (NfL) levels, a biomarker for neurodegeneration, were further elevated in the Grn/Trem2 KO cerebrospinal fluid (CSF). These findings suggest that TREM2-dependent microglia hyperactivation in models of GRN deficiency does not promote neurotoxicity, but rather neuroprotection.
    Keywords:  frontotemporal lobar degeneration; lysosomes; microglia; neurodegeneration; progranulin
    DOI:  https://doi.org/10.15252/embj.2021109108
  16. J Clin Invest. 2022 Jan 13. pii: e146286. [Epub ahead of print]
    AAV9/MFSD8 gene therapy is effective in preclinical models of neuronal ceroid lipofuscinosis type 7 disease.
    Xin Chen, Thomas Dong, Yuhui Hu, Frances C Shaffo, Nandkishore R Belur, Joseph R Mazzulli, Steven J Gray.
      Neuronal ceroid lipofuscinosis type 7 (CLN7) disease is a lysosomal storage disease caused by mutations in the facilitator superfamily domain containing 8 (MFSD8) gene, which encodes a membrane-bound lysosomal protein MFSD8. To test the effectiveness and safety of adeno-associated viral (AAV) gene therapy, an in vitro study demonstrated that AAV2/MFSD8 dose-dependently rescued lysosomal function in fibroblasts from a CLN7 patient. An in vivo efficacy study using intrathecal administration of AAV9/MFSD8 to Mfsd8-/- mice at postnatal day (p)7-10 or p120 with high or low dose led to clear age- and dose-dependent effects. A high dose of AAV9/MFSD8 at p7-10 resulted in widespread MFSD8 mRNA expression, tendency of amelioration of subunit c of mitochondrial ATP synthase accumulation and glial fibrillary acidic protein immunoreactivity, normalization of impaired behaviors, doubled median lifespan, and extended normal body weight gain. In vivo safety studies in rodents concluded that intrathecal administration of AAV9/MFSD8 was safe and well-tolerated. In summary, these results demonstrated that the AAV9/MFSD8 vector is both effective and safe in preclinical models. Investigational New Drug application #19766 to initiate a Phase I intrathecal gene transfer trial for AAV9/MFSD8 was approved by the US FDA and the trial is enrolling CLN7 patients at Children's Health in Dallas, TX in collaboration with UTSW Medical Center (clinicaltrials.gov NCT04737460).
    Keywords:  Gene therapy; Genetic diseases; Neurodegeneration; Neuroscience
    DOI:  https://doi.org/10.1172/JCI146286
  17. Autophagy. 2022 Jan 09. 1-2
    RABEP1/Rabaptin5: a link between autophagy and early endosome homeostasis.
    Valentina Millarte, Martin Spiess.
      Selective autophagy of damaged organelles assures maintenance of cellular homeostasis in eukaryotes. While the mechanisms by which cells selectively remove dysfunctional mitochondria, lysosomes, endoplasmic reticulum and other organelles has been well characterized, little is known about specific autophagy of damaged early endosomes. In our recent study, we uncovered a new role for RABEP1/Rabaptin5, a long-established regulator of early endosome function, in targeting the autophagy machinery to early endosomes damaged by chloroquine or by internalized Salmonella via interaction with RB1CC1/FIP200 and ATG16L1.
    Keywords:  ATG16L1; FIP200; Rabaptin5; Salmonella; autophagy; early endosome
    DOI:  https://doi.org/10.1080/15548627.2021.2021497
  18. Sci Rep. 2022 Jan 12. 12(1): 596
    Annexin A6 and NPC1 regulate LDL-inducible cell migration and distribution of focal adhesions.
    Jaimy Jose, Monira Hoque, Johanna Engel, Syed S Beevi, Mohamed Wahba, Mariya Ilieva Georgieva, Kendelle J Murphy, William E Hughes, Blake J Cochran, Albert Lu, Francesc Tebar, Andrew J Hoy, Paul Timpson, Kerry-Anne Rye, Carlos Enrich, Carles Rentero, Thomas Grewal.
      Cholesterol is considered indispensable for cell motility, but how physiological cholesterol pools enable cells to move forward remains to be clarified. The majority of cells obtain cholesterol from the uptake of Low-Density lipoproteins (LDL) and here we demonstrate that LDL stimulates A431 squamous epithelial carcinoma and Chinese hamster ovary (CHO) cell migration and invasion. LDL also potentiated epidermal growth factor (EGF) -stimulated A431 cell migration as well as A431 invasion in 3-dimensional environments, using organotypic assays. Blocking cholesterol export from late endosomes (LE), using Niemann Pick Type C1 (NPC1) mutant cells, pharmacological NPC1 inhibition or overexpression of the annexin A6 (AnxA6) scaffold protein, compromised LDL-inducible migration and invasion. Nevertheless, NPC1 mutant cells established focal adhesions (FA) that contain activated focal adhesion kinase (pY397FAK, pY861FAK), vinculin and paxillin. Compared to controls, NPC1 mutants display increased FA numbers throughout the cell body, but lack LDL-inducible FA formation at cell edges. Strikingly, AnxA6 depletion in NPC1 mutant cells, which restores late endosomal cholesterol export in these cells, increases their cell motility and association of the cholesterol biosensor D4H with active FAK at cell edges, indicating that AnxA6-regulated transport routes contribute to cholesterol delivery to FA structures, thereby improving NPC1 mutant cell migratory behaviour.
    DOI:  https://doi.org/10.1038/s41598-021-04584-y
  19. Commun Biol. 2022 Jan 10. 5(1): 5
    JIP3 links lysosome transport to regulation of multiple components of the axonal cytoskeleton.
    N M Rafiq, L L Lyons, S Gowrishankar, P De Camilli, S M Ferguson.
      Lysosome axonal transport is important for the clearance of cargoes sequestered by the endocytic and autophagic pathways. Building on observations that mutations in the JIP3 (MAPK8IP3) gene result in lysosome-filled axonal swellings, we analyzed the impact of JIP3 depletion on the cytoskeleton of human neurons. Dynamic focal lysosome accumulations were accompanied by disruption of the axonal periodic scaffold (spectrin, F-actin and myosin II) throughout each affected axon. Additionally, axonal microtubule organization was locally disrupted at each lysosome-filled swelling. This local axonal microtubule disorganization was accompanied by accumulations of both F-actin and myosin II. These results indicate that transport of axonal lysosomes is functionally interconnected with mechanisms that control the organization and maintenance of the axonal cytoskeleton. They have potential relevance to human neurological disease arising from JIP3 mutations as well as for neurodegenerative diseases associated with the focal accumulations of lysosomes within axonal swellings such as Alzheimer's disease.
    DOI:  https://doi.org/10.1038/s42003-021-02945-x
  20. ACS Appl Bio Mater. 2020 Feb 17. 3(2): 977-985
    Inhibition of Autophagy via Lysosomal Impairment Enhances Cytotoxicity of Fullerenol under Starvation Condition.
    Liyun Yang, Siyu Hua, Junpeng Fan, Zhiqiang Zhou, Guanchao Wang, Fenglei Jiang, Zhixiong Xie, Qi Xiao, Yi Liu.
      Autophagy is well-known as a common cellular response to nanomaterials. As one of the most comprehensively studied carbon-based nanomaterials, fullerene and its derivatives have been reported to bring about autophagic features in various cell lines, but little is known about the role of fullerenol (C60(OH)44) on the modulation of autophagy in human gastric tumor cell line SGC-7901. Fullerenol treatment led to the accumulation of autophagosomes, as evidenced by the increased fluorescent intensity of monodansylcadaverine (MDC) staining cells, an elevated level of LC3 protein, and the observation of auotphagosomes in cytoplasm. Subsequent results of the p62 level demonstrated that the accumulation of autophagosomes resulted from the blockade of autophagic flux rather than the activation of autophagy. Fullerenol disrupted autophagic flux by impairing lysosomal function, including lysosome membrane permeabilization (LMP), alkaline of lysosomes, and reduced activity of capthesin B. Interestingly, fullerenol treatment was noncytotoxic under a nutrient-rich condition. When serum was deprived, cytotoxicity occurred in a concentration- and time-dependent manner, along with massive vacuoles in cytoplasm, a large amount of ROS generation, and finally cell death, which can be ascribed to the disruption of essential autophagy in cells. Taken together, understanding this autophagy-lysosome pathway will shed light on the potential anticancer application of fullerenol.
    Keywords:  autophagy; cytotoxicity; fullerenol; lysosomal dysfunction; starvation
    DOI:  https://doi.org/10.1021/acsabm.9b01001
  21. Cell Metab. 2022 Jan 07. pii: S1550-4131(21)00636-7. [Epub ahead of print]
    Mosaic dysfunction of mitophagy in mitochondrial muscle disease.
    Takayuki Mito, Amy E Vincent, Julie Faitg, Robert W Taylor, Nahid A Khan, Thomas G McWilliams, Anu Suomalainen.
      Mitophagy is a quality control mechanism that eliminates damaged mitochondria, yet its significance in mammalian pathophysiology and aging has remained unclear. Here, we report that mitophagy contributes to mitochondrial dysfunction in skeletal muscle of aged mice and human patients. The early disease stage is characterized by muscle fibers with central nuclei, with enhanced mitophagy around these nuclei. However, progressive mitochondrial dysfunction halts mitophagy and disrupts lysosomal homeostasis. Interestingly, activated or halted mitophagy occur in a mosaic manner even in adjacent muscle fibers, indicating cell-autonomous regulation. Rapamycin restores mitochondrial turnover, indicating mTOR-dependence of mitochondrial recycling in advanced disease stage. Our evidence suggests that (1) mitophagy is a hallmark of age-related mitochondrial pathology in mammalian muscle, (2) mosaic halting of mitophagy is a mechanism explaining mosaic respiratory chain deficiency and accumulation of pathogenic mtDNA variants in adult-onset mitochondrial diseases and normal aging, and (3) augmenting mitophagy is a promising therapeutic approach for muscle mitochondrial dysfunction.
    Keywords:  SBFSEM; centrally nucleated fibers; lysosome; mito-QC; mitochondrial disease; mitochondrial myopathy; mitophagy; patient; ragged-red fibers
    DOI:  https://doi.org/10.1016/j.cmet.2021.12.017
  22. JIMD Rep. 2022 Jan;63(1): 50-65
    Chronic lithium administration in a mouse model for Krabbe disease.
    Ambra Del Grosso, Gabriele Parlanti, Lucia Angella, Nadia Giordano, Ilaria Tonazzini, Elisa Ottalagana, Sara Carpi, Roberto Maria Pellegrino, Husam B R Alabed, Carla Emiliani, Matteo Caleo, Marco Cecchini.
      Krabbe disease (KD; or globoid cell leukodystrophy) is an autosomal recessive lysosomal storage disorder caused by deficiency of the galactosylceramidase (GALC) enzyme. No cure is currently available for KD. Clinical applied treatments are supportive only. Recently, we demonstrated that two differently acting autophagy inducers (lithium and rapamycin) can improve some KD hallmarks in-vitro, laying the foundation for their in-vivo pre-clinical testing. Here, we test lithium carbonate in-vivo, in the spontaneous mouse model for KD, the Twitcher (TWI) mouse. The drug is administered ad libitum via drinking water (600 mg/L) starting from post natal day 20. We longitudinally monitor the mouse motor performance through the grip strength, the hanging wire and the rotarod tests, and a set of biochemical parameters related to the KD pathogenesis [i.e., GALC enzymatic activity, psychosine (PSY) accumulation and astrogliosis]. Additionally, we investigate the expression of some crucial markers related to the two pathways that could be altered by lithium: the autophagy and the β-catenin-dependent pathways. Results demonstrate that lithium has not a significant rescue effect on the TWI phenotype, although it can slightly and transiently improves muscle strength. We also show that lithium, with this administration protocol, is unable to stimulate autophagy in the TWI mice central nervous system, whereas results suggest that it can restore the β-catenin activation status in the TWI sciatic nerve. Overall, these data provide intriguing inputs for further evaluations of lithium treatment in TWI mice.
    Keywords:  Krabbe; Twitcher; autophagy; globoid cell leukodystrophy; lithium; psychosine
    DOI:  https://doi.org/10.1002/jmd2.12258
  23. Commun Biol. 2022 Jan 12. 5(1): 47
    Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins.
    Cliff J Luke, Stephanie Markovina, Misty Good, Ira E Wight, Brian J Thomas, John M Linneman, Wyatt E Lanik, Olga Koroleva, Maggie R Coffman, Mark T Miedel, Qingqing Gong, Arlise Andress, Marlene Campos Guerrero, Songyan Wang, LiYun Chen, Wandy L Beatty, Kelsey N Hausmann, Frances V White, James A J Fitzpatrick, Anthony Orvedahl, Stephen C Pak, Gary A Silverman.
      Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans (C. elegans) null for a cysteine protease inhibitor, srp-6, undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3, respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans, this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors.
    DOI:  https://doi.org/10.1038/s42003-021-02953-x
  24. J Gene Med. 2022 Jan 14. e3410
    Brain and visceral gene editing of mucopolysaccharidosis I mice by nasal delivery of the CRISPR/Cas9 system.
    Luisa Natalia Pimentel Vera, Roselena Silvestri Schuh, Flavia Nathielly Silveira Fachel, Edina Poletto, Eduarda Piovesan, Francyne Kubaski, Eduarda Couto, Bruna Brum, Graziella Rodrigues, Hallana Souza, Roberto Giugliani, Ursula Matte, Guilherme Baldo, Helder F Teixeira.
       INTRODUCTION: Mucopolysaccharidosis type I (MPS I) is an inherited disease caused by deficiency of the enzyme alpha-L-iduronidase (IDUA). MPS I affects several tissues, including the brain, leading to cognitive impairment in the severe form of the disease. Currently available treatments do not reach the brain. Therefore, in this study, we performed nasal administration (NA) of liposomal complexes carrying two plasmids encoding for the CRISPR/Cas9 system and for the IDUA gene targeting the ROSA26 locus, aiming at brain delivery in MPS I mice.
    METHODS: Liposomes were prepared by microfluidization and the plasmids were complexed to the formulations by adsorption. Physicochemical characterization of the formulations and complexes, in vitro permeation, and mucoadhesion in porcine nasal mucosa (PNM) were assessed. We performed NA repeatedly for 30 days in young MPS I mice, which were euthanized at 6 months of age after performing behavioral tasks, and biochemical and molecular aspects were evaluated.
    RESULTS: Monodisperse mucoadhesive complexes around 110nm, which are able to efficiently permeate the PNM. In animals the treatment led to a modest increase in IDUA activity in the lung, heart and brain areas, with reduction of glycosaminoglycan (GAG) levels in serum, urine, tissues and brain cortex. Furthermore, treated mice showed improvement in behavioral tests, suggesting prevention of the cognitive damage.
    CONCLUSION: Non-viral gene editing performed through nasal route represents a potential therapeutic alternative for the somatic and neurologic symptoms of MPS I and possibly to other neurological disorders.
    Keywords:  CRISPR/Cas; Genome editing; Hurler syndrome; Liposome; Lysosomal storage disease; Mucopolysaccharidosis type I; Nonviral vector
    DOI:  https://doi.org/10.1002/jgm.3410
  25. Cell Rep. 2022 Jan 11. pii: S2211-1247(21)01487-X. [Epub ahead of print]38(2): 110009
    CDC42 controlled apical-basal polarity regulates intestinal stem cell to transit amplifying cell fate transition via YAP-EGF-mTOR signaling.
    Zheng Zhang, Feng Zhang, Ashley Kuenzi Davis, Mei Xin, Gerd Walz, Weidong Tian, Yi Zheng.
      Epithelial polarity is controlled by a polarity machinery that includes Rho GTPase CDC42 and Scribble/PAR. By using intestinal stem cell (ISC)-specific deletion of CDC42 in olfactomedin-4 (Olfm4)-internal ribosome entry site (IRES)-EGFP/CreERT2;CDC42flox/flox mice, we find that CDC42 loss initiated in the ISCs causes a drastic hyperproliferation of transit amplifying (TA) cells and disrupts epithelial polarity. CDC42-null crypts display expanded TA cell and diminished ISC populations, accompanied by elevated Hippo signaling via YAP/TAZ-Ereg (yes-associated protein/WW domain-containing transcription regulator protein 1-epiregulin) and mechanistic target of rapamycin (mTOR) activation, independent from canonical Wnt signaling. YAP/TAZ conditional knockout (KO) restores the balance of ISC/TA cell populations and crypt proliferation but does not rescue the polarity in CDC42-null small intestine. mTOR or epidermal growth factor receptor (EGFR) inhibitor treatment of CDC42 KO mice exhibits similar rescuing effects without affecting YAP/TAZ signaling. Inducible ablation of Scribble in intestinal epithelial cells mimics that of CDC42 KO defects, including crypt hyperplasia and Hippo signaling activation. Mammalian epithelial polarity regulates ISC/TA cell fate and proliferation via a Hippo-Ereg-mTOR cascade.
    Keywords:  Cdc42; Hippo signaling; cell fate; intestinal stem cells; mTOR signaling; mouse model; polarity
    DOI:  https://doi.org/10.1016/j.celrep.2021.110009
  26. J Exp Clin Cancer Res. 2022 Jan 10. 41(1): 18
    RNA binding protein HuD promotes autophagy and tumor stress survival by suppressing mTORC1 activity and augmenting ARL6IP1 levels.
    Kausik Bishayee, Khadija Habib, Uddin Md Nazim, Jieun Kang, Aniko Szabo, Sung-Oh Huh, Ali Sadra.
       BACKGROUND: Neuronal-origin HuD (ELAVL4) is an RNA binding protein overexpressed in neuroblastoma (NB) and certain other cancers. The RNA targets of this RNA binding protein in neuroblastoma cells and their role in promoting cancer survival have been unexplored. In the study of modulators of mTORC1 activity under the conditions of optimal cell growth and starvation, the role of HuD and its two substrates were studied.
    METHODS: RNA immunoprecipitation/sequencing (RIP-SEQ) coupled with quantitative real-time PCR were used to identify substrates of HuD in NB cells. Validation of the two RNA targets of HuD was via reverse capture of HuD by synthetic RNA oligoes from cell lysates and binding of RNA to recombinant forms of HuD in the cell and outside of the cell. Further analysis was via RNA transcriptome analysis of HuD silencing in the test cells.
    RESULTS: In response to stress, HuD was found to dampen mTORC1 activity and allow the cell to upregulate its autophagy levels by suppressing mTORC1 activity. Among mRNA substrates regulated cell-wide by HuD, GRB-10 and ARL6IP1 were found to carry out critical functions for survival of the cells under stress. GRB-10 was involved in blocking mTORC1 activity by disrupting Raptor-mTOR kinase interaction. Reduced mTORC1 activity allowed lifting of autophagy levels in the cells required for increased survival. In addition, ARL6IP1, an apoptotic regulator in the ER membrane, was found to promote cell survival by negative regulation of apoptosis. As a therapeutic target, knockdown of HuD in two xenograft models of NB led to a block in tumor growth, confirming its importance for viability of the tumor cells. Cell-wide RNA messages of these two HuD substrates and HuD and mTORC1 marker of activity significantly correlated in NB patient populations and in mouse xenografts.
    CONCLUSIONS: HuD is seen as a novel means of promoting stress survival in this cancer type by downregulating mTORC1 activity and negatively regulating apoptosis.
    Keywords:  ARL6IP1; Cancer cell survival; ELAVL4; GRB-10; mTORC1
    DOI:  https://doi.org/10.1186/s13046-021-02203-2
  27. Biochim Biophys Acta Biomembr. 2022 Jan 11. pii: S0005-2736(21)00307-2. [Epub ahead of print] 183858
    Substrate-induced differential degradation and partitioning of the two tryptophan permeases Tat1 and Tat2 into eisosomes in Saccharomyces cerevisiae.
    Ryoga Ishii, Ayu Fukui, Yuri Sakihama, Shoko Kitsukawa, Ayami Futami, Takahiro Mochizuki, Makoto Nagano, Jiro Toshima, Fumiyoshi Abe.
      Tryptophan is a relatively rare amino acid whose influx is strictly controlled to meet cellular demands. The yeast Saccharomyces cerevisiae has two tryptophan permeases, namely Tat1 (low-affinity type) and Tat2 (high-affinity type). These permeases are differentially regulated through ubiquitination based on inducible conditions and dependence on arrestin-related trafficking adaptors, although the physiological significance of their degradation remain unclear. Here, we demonstrated that Tat2 was rapidly degraded in an Rsp5-Bul1-dependent manner upon the addition of tryptophan, phenylalanine, or tyrosine, whereas Tat1 was unaffected. The expression of the ubiquitination-deficient variant Tat25K>R led to a reduction in cell yield at 4 μg/mL tryptophan, suggesting the occurrence of an uncontrolled, excessive consumption of tryptophan at low tryptophan concentrations. Eisosomes are membrane furrows that are thought to be storage compartments for some nutrient permeases. Tryptophan addition caused rapid Tat2 dissociation from eisosomes, whereas Tat1 distribution was unaffected. The 5 K > R mutation had no marked effect on Tat2 dissociation, suggesting that dissociation is independent of ubiquitination. Interestingly, the D74R mutation, which was created within the N-terminal acidic patch, stabilized Tat2 while reducing the degree of partitioning into eisosomes. Moreover, the hyperactive I285V mutation in Tat2, which increases Vmax/Km for tryptophan import by 2-fold, reduced the degree of segregation into eisosomes. Our findings illustrate the coordinated activity of Tat1 and Tat2 in the regulation of tryptophan transport at various tryptophan concentrations and suggest the positive role of substrates in inducing a conformational transition in Tat2, resulting in its dissociation from eisosomes and subsequent ubiquitination-dependent degradation.
    Keywords:  Degradation; Eisosomes; Pil1; Rsp5-Bul1 complex; Saccharomyces cerevisiae; Tat1; Tat2; Tryptophan permease
    DOI:  https://doi.org/10.1016/j.bbamem.2021.183858
  28. iScience. 2022 Jan 21. 25(1): 103645
    Translational and post-translational control of human naïve versus primed pluripotency.
    Cheng Chen, Xiaobing Zhang, Yisha Wang, Xinyu Chen, Wenjie Chen, Songsong Dan, Shiqi She, Weiwei Hu, Jie Dai, Jianwen Hu, Qingyi Cao, Qianyu Liu, Yinghua Huang, Baoming Qin, Bo Kang, Ying-Jie Wang.
      Deciphering the regulatory network for human naive and primed pluripotency is of fundamental theoretical and applicable significance. Here, by combining quantitative proteomics, phosphoproteomics, and acetylproteomics analyses, we revealed RNA processing and translation as the most differentially regulated processes between naive and primed human embryonic stem cells (hESCs). Although glycolytic primed hESCs rely predominantly on the eukaryotic initiation factor 4E (eIF4E)-mediated cap-dependent pathway for protein translation, naive hESCs with reduced mammalian target of rapamycin complex (mTORC1) activity are more tolerant to eIF4E inhibition, and their bivalent metabolism allows for translating selective mRNAs via both eIF4E-dependent and eIF4E-independent/eIF4A2-dependent pathways to form a more compact naive proteome. Globally up-regulated proteostasis and down-regulated post-translational modifications help to further refine the naive proteome that is compatible with the more rapid cycling of naive hESCs, where CDK1 plays an indispensable coordinative role. These findings may assist in better understanding the unrestricted lineage potential of naive hESCs and in further optimizing conditions for future clinical applications.
    Keywords:  Molecular biology; Proteomics; Stem cells research
    DOI:  https://doi.org/10.1016/j.isci.2021.103645
  29. ACS Appl Bio Mater. 2021 Dec 09.
    In Cellulo Crystallization of Human Neuraminidase 1 and Biological Roles of N-Glycans.
    Jun Tsukimoto, Yoshie Takeuchi, Yuto Horii, Toshiki Iniwa, Yuri Fukushi, So-Ichiro Nishioka, Kohji Itoh.
      Human neuraminidase 1 (NEU1) is a lysosomal glycosidase that cleaves the terminal sialic acids of sialylglycoconjugates. NEU1 is biosynthesized in the endoplasmic reticulum (ER) lumen as an N-glycosylated protein. NEU1 also associates with cathepsin A (CTSA) in ER, migrates to lysosomes, and exerts catalytic activity. Extraordinary in cellulo crystallization of NEU1 protein in ER despite carrying three N-glycans per molecule at N186, N343, and N352, respectively, were observed when the single human NEU1 gene was overexpressed in mammalian cells. In this study, we first purified the NEU1 from the isolated crystals produced by the HEK293 NEU1-KO cell transiently overexpressing the normal NEU1 and found that the N-glycans were high-mannose or complex types carrying terminal sialic acids. The result suggests that a part of NEU1 crystals were formed or transported to the Golgi apparatus. Second, we compared the effects of single amino acid substitution at the N-sequons, including N186Q, N343Q, and N352Q, each one N-glycan reduction from one NEU1 molecule. We demonstrated that N186Q mutant protein with low enzyme activity and formed a few amounts of smaller crystals. The N343Q mutant exhibited half of the normal intracellular activity, but the numbers and sizes of crystals were almost the same as those of normal NEU1. The N352Q mutant exhibited almost the same activity as the normal enzyme. The numbers of the N352Q crystals were smaller than those of normal NEU1. According to these findings, the N186Q NEU1 protein should have lower stability in ER due to abnormal folding. The second N-glycan at the N343-sequon has little effect on self-aggregation of NEU1. The third N-glycan at the N352-sequon contributes to the self-aggregation of NEU1. We also demonstrated that the three NEU1 mutants associate with the relatively excessive CTSA and migrate to lysosomes.
    Keywords:  N-glycans; human lysosomal sialidase; human neuraminidase; in cellulo glycoprotein crystals; lysosomal cathepsin A; sialic acid
    DOI:  https://doi.org/10.1021/acsabm.1c01043
  30. Toxicol Lett. 2022 Jan 06. pii: S0378-4274(21)00926-7. [Epub ahead of print]
    Methylmercury induces lysosomal membrane permeabilization through JNK-activated Bax lysosomal translocation in neuronal cells.
    Lin Tianji, Huang Dingbang, Chen Xiao, Meng Xiaojing, Zou Fei, Wang Bin.
      MeHg, an environmental toxicant, is highly toxic to the central nervous system. Recent studies have reported that LMP is an important way in the lysosomal damage. However, the role and molecular mechanism of LMP in MeHg-induced neurotoxicity remain unknown. To study MeHg-induced LMP, we used 10µM MeHg to treat SH-SY5Y cells and 2µM MeHg to treat rat cerebral cortical neurons. Acridine orange (AO) staining and analysis of cathepsin B (CTSB) release were used to determine LMP. We found that MeHg reduced red AO fluorescence and induced CTSB release from lysosomes to the cytoplasm in a time-dependent manner. Moreover, pretreatment with the CTSB inhibitor alleviated cytotoxicity in neuronal cells. These results indicate MeHg induces LMP and subsequent CTSB-dependent cytotoxicity in neuronal cells. Bax is a pore-forming protein, which is involved in mitochondrial outer membrane permeabilization. Intriguingly, we demonstrated that MeHg induced Bax to translocate to lysosomes by using immunofluorescence and Western blot analysis of subcellular fractions. Furthermore, downregulating Bax expression suppressed MeHg-induced LMP. Bax subcellular localization is regulated by protein interaction with the cytoplasmic 14-3-3. Our previous study demonstrated that JNK participated in neurotoxicity through regulating protein interaction. In the current study, we showed that JNK dissociated Bax-14-3-3 complex to facilitate Bax lysosomal translocation. Finally, inhibition of the JNK/Bax pathway could alleviate MeHg-induced cytotoxicity in neuronal cells. The present study implies that inhibiting lysosomal damage (LMP)-related signaling might alleviate MeHg neurotoxicity.
    Keywords:  Bax; JNK; Lysosomal membrane permeabilization; Lysosomal translocation; Methylmercury
    DOI:  https://doi.org/10.1016/j.toxlet.2021.12.021
  31. Prog Lipid Res. 2022 Jan 06. pii: S0163-7827(22)00001-7. [Epub ahead of print] 101146
    Coordination of inter-organelle communication and lipid fluxes by OSBP-related proteins.
    Amita Arora, Juuso H Taskinen, Vesa M Olkkonen.
      Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute one of the largest families of lipid-binding/transfer proteins (LTPs) in eukaryotes. The current view is that many of them mediate inter-organelle lipid transfer over membrane contact sites (MCS). The transfer occurs in several cases in a 'counter-current' fashion: A lipid such as cholesterol or phosphatidylserine (PS) is transferred against its concentration gradient driven by transport of a phosphoinositide in the opposite direction. In this way ORPs are envisioned to maintain the distinct organelle lipid compositions, with impacts on multiple organelle functions. However, the functions of ORPs extend beyond lipid homeostasis to regulation of processes such as cell survival, proliferation and migration. Important expanding areas of mammalian ORP research include their roles in viral and bacterial infections, cancers, and neuronal function. The yeast OSBP homologue (Osh) proteins execute multifaceted functions in sterol and glycerophospholipid homeostasis, post-Golgi vesicle transport, phosphatidylinositol-4-phosphate, sphingolipid and target of rapamycin (TOR) signalling, and cell cycle control. These observations identify ORPs as lipid transporters and coordinators of signals with an unforeseen variety of cellular processes. Understanding their activities not only enlightens the biology of the living cell but also allows their employment as targets of new therapeutic approaches for disease.
    Keywords:  Cell signalling; Lipid metabolism; Lipid transport; Membrane contact site; ORP; OSBPL
    DOI:  https://doi.org/10.1016/j.plipres.2022.101146
  32. Trends Biochem Sci. 2022 Jan 07. pii: S0968-0004(21)00274-7. [Epub ahead of print]
    Structural advances in sterol-sensing domain-containing proteins.
    Xuelan Wu, Renhong Yan, Pingping Cao, Hongwu Qian, Nieng Yan.
      The sterol-sensing domain (SSD) is present in several membrane proteins that function in cholesterol metabolism, transport, and signaling. Recent progress in structural studies of SSD-containing proteins, such as sterol regulatory element-binding protein (SREBP)-cleavage activating protein (Scap), Patched, Niemann-Pick disease type C1 (NPC1), and related proteins, reveals a conserved core that is essential for their sterol-dependent functions. This domain, by its name, 'senses' the presence of sterol substrates through interactions and may modulate protein behaviors with changing sterol levels. We summarize recent advances in structural and mechanistic investigations of these proteins and propose to divide them to two classes: M for 'moderator' proteins that regulate sterol metabolism in response to membrane sterol levels, and T for 'transporter' proteins that harbor inner tunnels for cargo trafficking across cellular membranes.
    Keywords:  Insig; Niemann-Pick disease type C1 (NPC1); Patched; Scap; cholesterol metabolism; cryo-EM
    DOI:  https://doi.org/10.1016/j.tibs.2021.12.005
  33. Genes Dis. 2022 Jan;9(1): 187-200
    Tsc2 mutation induces renal tubular cell nonautonomous disease.
    Prashant Kumar, Fahad Zadjali, Ying Yao, Daniel Johnson, Brian Siroky, Aristotelis Astrinidis, Peter Vogel, Kenneth W Gross, John J Bissler.
      TSC renal cystic disease is poorly understood and has no approved treatment. In a new principal cell-targeted murine model of Tsc cystic disease, the renal cystic epithelium is mostly composed of type A intercalated cells with an intact Tsc2 gene confirmed by sequencing, although these cells exhibit a Tsc-mutant disease phenotype. We used a newly derived targeted murine model in lineage tracing and extracellular vesicle (EV) characterization experiments and a cell culture model in EV characterization and cellular induction experiments to understand TSC cystogenesis. Using lineage tracing experiments, we found principal cells undergo clonal expansion but contribute very few cells to the cyst. We determined that cystic kidneys contain more interstitial EVs than noncystic kidneys, excrete fewer EVs in urine, and contain EVs in cyst fluid. Moreover, the loss of Tsc2 gene in EV-producing cells greatly changes the effect of EVs on renal tubular epithelium, such that the epithelium develops increased secretory and proliferative pathway activity. We demonstate that the mTORC1 pathway activity is independent form the EV production, and that the EV effects for a single cell line can vary significantly. TSC cystogenesis involves significant contribution from genetically intact cells conscripted to the mutant phenotype by mutant cell derived EVs.
    Keywords:  Cell nonautonomous trait; Polycystic kidney disease; Renalcystogenesis; Tuberous sclerosis complex
    DOI:  https://doi.org/10.1016/j.gendis.2021.03.010
  34. Cells. 2021 Dec 30. pii: 119. [Epub ahead of print]11(1):
    Selectivity of mTOR-Phosphatidic Acid Interactions Is Driven by Acyl Chain Structure and Cholesterol.
    Jolanta Żelasko, Aleksander Czogalla.
      The need to gain insights into the molecular details of peripheral membrane proteins' specificity towards phosphatidic acid (PA) is undeniable. The variety of PA species classified in terms of acyl chain length and saturation translates into a complicated, enigmatic network of functional effects that exert a critical influence on cell physiology. As a consequence, numerous studies on the importance of phosphatidic acid in human diseases have been conducted in recent years. One of the key proteins in this context is mTOR, considered to be the most important cellular sensor of essential nutrients while regulating cell proliferation, and which also appears to require PA to build stable and active complexes. Here, we investigated the specific recognition of three physiologically important PA species by the mTOR FRB domain in the presence or absence of cholesterol in targeted membranes. Using a broad range of methods based on model lipid membrane systems, we elucidated how the length and saturation of PA acyl chains influence specific binding of the mTOR FRB domain to the membrane. We also discovered that cholesterol exerts a strong modulatory effect on PA-FRB recognition. Our data provide insight into the molecular details of some physiological effects reported previously and reveal novel mechanisms of fine-tuning the signaling cascades dependent on PA.
    Keywords:  BLI; GUV; bio-layer interferometry; giant unilamellar vesicles; lipid signaling; liposomes; mTOR; phosphatidic acid; protein-lipid interactions
    DOI:  https://doi.org/10.3390/cells11010119
  35. Cells. 2021 Dec 21. pii: 6. [Epub ahead of print]11(1):
    In Vitro and In Vivo Models to Study Nephropathic Cystinosis.
    Pang Yuk Cheung, Patrick T Harrison, Alan J Davidson, Jennifer A Hollywood.
      The development over the past 50 years of a variety of cell lines and animal models has provided valuable tools to understand the pathophysiology of nephropathic cystinosis. Primary cultures from patient biopsies have been instrumental in determining the primary cause of cystine accumulation in the lysosomes. Immortalised cell lines have been established using different gene constructs and have revealed a wealth of knowledge concerning the molecular mechanisms that underlie cystinosis. More recently, the generation of induced pluripotent stem cells, kidney organoids and tubuloids have helped bridge the gap between in vitro and in vivo model systems. The development of genetically modified mice and rats have made it possible to explore the cystinotic phenotype in an in vivo setting. All of these models have helped shape our understanding of cystinosis and have led to the conclusion that cystine accumulation is not the only pathology that needs targeting in this multisystemic disease. This review provides an overview of the in vitro and in vivo models available to study cystinosis, how well they recapitulate the disease phenotype, and their limitations.
    Keywords:  cell and animal models; cystinosis; lysosomal storage disease
    DOI:  https://doi.org/10.3390/cells11010006
  36. Nat Commun. 2022 Jan 14. 13(1): 318
    Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice.
    Barbara Spix, Elisabeth S Butz, Cheng-Chang Chen, Anna Scotto Rosato, Rachel Tang, Aicha Jeridi, Veronika Kudrina, Eva Plesch, Philipp Wartenberg, Elisabeth Arlt, Daria Briukhovetska, Meshal Ansari, Gizem Günes Günsel, Thomas M Conlon, Amanda Wyatt, Sandra Wetzel, Daniel Teupser, Lesca M Holdt, Fabien Ectors, Ingrid Boekhoff, Ulrich Boehm, Jaime García-Añoveros, Paul Saftig, Martin Giera, Sebastian Kobold, Herbert B Schiller, Susanna Zierler, Thomas Gudermann, Christian Wahl-Schott, Franz Bracher, Ali Önder Yildirim, Martin Biel, Christian Grimm.
      Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease.
    DOI:  https://doi.org/10.1038/s41467-021-27860-x
  37. Autophagy. 2022 Jan 14. 1-2
    Plant virus infection disrupts vacuolar acidification and autophagic degradation for the effective infection.
    Meng Yang, Yan Wang, Dawei Li, Yule Liu.
      Vacuoles are the largest compartments in plant cells and are involved in plant development and response to abiotic and biotic stresses. Vacuolar acidification is essential for vacuoles in various physiological functions. However, its role in plant defense, and whether and how pathogens affect vacuolar acidification to promote infection have never been reported. In this autophagy punctum, we discuss our recent findings about how plant viruses suppress vacuolar acidification and the degradation of autophagic bodies by directly interacting with a component of the V-ATPase to promote virus infection.
    Keywords:  Autophagic degradation; V-ATPase; autophagy; defense; vacuolar acidification; virus
    DOI:  https://doi.org/10.1080/15548627.2022.2027194
  38. Commun Biol. 2022 Jan 12. 5(1): 46
    SERPINB3 (SCCA1) inhibits cathepsin L and lysoptosis, protecting cervical cancer cells from chemoradiation.
    Songyan Wang, Cliff J Luke, Stephen C Pak, Victoria Shi, Liyun Chen, Jonathan Moore, Arlise P Andress, Kay Jayachandran, Jin Zhang, Yi Huang, Marina Platik, Anthony A Apicelli, Julie K Schwarz, Perry W Grigsby, Gary A Silverman, Stephanie Markovina.
      The endogenous lysosomal cysteine protease inhibitor SERPINB3 (squamous cell carcinoma antigen 1, SCCA1) is elevated in patients with cervical cancer and other malignancies. High serum SERPINB3 is prognostic for recurrence and death following chemoradiation therapy. Cervical cancer cells genetically lacking SERPINB3 are more sensitive to ionizing radiation (IR), suggesting this protease inhibitor plays a role in therapeutic response. Here we demonstrate that SERPINB3-deficient cells have enhanced sensitivity to IR-induced cell death. Knock out of SERPINB3 sensitizes cells to a greater extent than cisplatin, the current standard of care. IR in SERPINB3 deficient cervical carcinoma cells induces predominantly necrotic cell death, with biochemical and cellular features of lysoptosis. Rescue with wild-type SERPINB3 or a reactive site loop mutant indicates that protease inhibitory activity is required to protect cervical tumor cells from radiation-induced death. Transcriptomics analysis of primary cervix tumor samples and genetic knock out demonstrates a role for the lysosomal protease cathepsin L in radiation-induced cell death in SERPINB3 knock-out cells. These data support targeting of SERPINB3 and lysoptosis to treat radioresistant cervical cancers.
    DOI:  https://doi.org/10.1038/s42003-021-02893-6
  39. Cell Death Differ. 2022 Jan 09.
    Site-specific ubiquitination of MLKL targets it to endosomes and targets Listeria and Yersinia to the lysosomes.
    Seongmin Yoon, Konstantin Bogdanov, David Wallach.
      Phosphorylation of the pseudokinase mixed lineage kinase domain-like protein (MLKL) by the protein kinase RIPK3 targets MLKL to the cell membrane, where it triggers necroptotic cell death. We report that conjugation of K63-linked polyubiquitin chains to distinct lysine residues in the N-terminal HeLo domain of phosphorylated MLKL (facilitated by the ubiquitin ligase ITCH that binds MLKL via a WW domain) targets MLKL instead to endosomes. This results in the release of phosphorylated MLKL within extracellular vesicles. It also prompts enhanced endosomal trafficking of intracellular bacteria such as Listeria monocytogenes and Yersinia enterocolitica to the lysosomes, resulting in decreased bacterial yield. Thus, MLKL can be directed by specific covalent modifications to differing subcellular sites, whence it signals either for cell death or for non-deadly defense mechanisms.
    DOI:  https://doi.org/10.1038/s41418-021-00924-7
  40. Elife. 2022 Jan 13. pii: e71256. [Epub ahead of print]11
    Disruption of PIKFYVE causes congenital cataract in human and zebrafish.
    Shaoyi Mei, Yi Wu, Yan Wang, Yubo Cui, Miao Zhang, Tong Zhang, Xiaosheng Huang, Sejie Yu, Tao Yu, Jun Zhao.
      Congenital cataract, an ocular disease predominantly occurring within the first decade of life, is one of the leading causes of blindness in children. However, the molecular mechanisms underlying the pathogenesis of congenital cataract remain incompletely defined. Through whole-exome sequencing of a Chinese family with congenital cataract, we identified a potential pathological variant (p.G1943E) in PIKFYVE, which is located in the PIP kinase domain of the PIKFYVE protein. We demonstrated that heterozygous/homozygous disruption of PIKFYVE kinase domain, instead of overexpression of PIKFYVEG1943E in zebrafish mimicked the cataract defect in human patients, suggesting that haploinsufficiency, rather than dominant-negative inhibition of PIKFYVE activity caused the disease. Phenotypical analysis of pikfyve zebrafish mutants revealed that loss of Pikfyve caused aberrant vacuolation (accumulation of Rab7+Lc3+ amphisomes) in lens cells, which was significantly alleviated by treatment with the V-ATPase inhibitor bafilomycin A1 (Baf-A1). Collectively, we identified PIKFYVE as a novel causative gene for congenital cataract and pinpointed the potential application of Baf-A1 for the treatment of congenital cataract caused by PIKFYVE deficiency.
    Keywords:  Baf-A1; PIKFYVE; congenital cataract; endosome; gene; genetics; genomics; mutation; zebrafish
    DOI:  https://doi.org/10.7554/eLife.71256
  41. Brain. 2022 Jan 06. pii: awac002. [Epub ahead of print]
    Prion-like α-synuclein pathology in the brain of infants with Krabbe disease.
    International DLB Genetics Consortium
      Krabbe disease is an infantile neurodegenerative disorder resulting from pathogenic variants in the GALC gene which causes accumulation of the toxic sphingolipid psychosine. GALC variants are also associated with Lewy body diseases, an umbrella term for age-associated neurodegenerative diseases in which the protein α-synuclein aggregates into Lewy bodies. To explore whether α-synuclein in Krabbe disease has pathological similarities to that in Lewy body disease, we performed an observational post-mortem study of Krabbe disease brain tissue (N = 4) compared to infant controls (N = 4) and identified widespread accumulations of α-synuclein. To determine whether α-synuclein in Krabbe disease brain displayed disease-associated pathogenic properties we evaluated its seeding capacity using the real-time quaking-induced conversion assay in two cases for which frozen tissue was available and strikingly identified aggregation into fibrils similar to those observed in Lewy body disease, confirming the prion-like capacity of Krabbe disease-derived α-synuclein. These observations constitute the first report of prion-like α-synuclein in the brain tissue of infants and challenge the putative view that α-synuclein pathology is merely an age-associated phenomenon, instead suggesting it results from alterations to biological pathways, such as sphingolipid metabolism. Our findings have important implications for understanding the mechanisms underlying Lewy body formation in Lewy body disease.
    Keywords:  Krabbe disease; Lewy body disease; sphingolipids; α-synuclein
    DOI:  https://doi.org/10.1093/brain/awac002
  42. J Neurodev Disord. 2022 Jan 15. 14(1): 8
    Down-regulation of the brain-specific cell-adhesion molecule contactin-3 in tuberous sclerosis complex during the early postnatal period.
    Anatoly Korotkov, Mark J Luinenburg, Alessia Romagnolo, Till S Zimmer, Jackelien van Scheppingen, Anika Bongaarts, Diede W M Broekaart, Jasper J Anink, Caroline Mijnsbergen, Floor E Jansen, Wim van Hecke, Wim G Spliet, Peter C van Rijen, Martha Feucht, Johannes A Hainfellner, Pavel Krsek, Josef Zamecnik, Peter B Crino, Katarzyna Kotulska, Lieven Lagae, Anna C Jansen, David J Kwiatkowski, Sergiusz Jozwiak, Paolo Curatolo, Angelika Mühlebner, Erwin A van Vliet, James D Mills, Eleonora Aronica.
       BACKGROUND: The genetic disorder tuberous sclerosis complex (TSC) is frequently accompanied by the development of neuropsychiatric disorders, including autism spectrum disorder and intellectual disability, with varying degrees of impairment. These co-morbidities in TSC have been linked to the structural brain abnormalities, such as cortical tubers, and recurrent epileptic seizures (in 70-80% cases). Previous transcriptomic analysis of cortical tubers revealed dysregulation of genes involved in cell adhesion in the brain, which may be associated with the neurodevelopmental deficits in TSC. In this study we aimed to investigate the expression of one of these genes - cell-adhesion molecule contactin-3.
    METHODS: Reverse transcription quantitative polymerase chain reaction for the contactin-3 gene (CNTN3) was performed in resected cortical tubers from TSC patients with drug-resistant epilepsy (n = 35, age range: 1-48 years) and compared to autopsy-derived cortical control tissue (n = 27, age range: 0-44 years), as well as by western blot analysis of contactin-3 (n = 7 vs n = 7, age range: 0-3 years for both TSC and controls) and immunohistochemistry (n = 5 TSC vs n = 4 controls). The expression of contactin-3 was further analyzed in fetal and postnatal control tissue by western blotting and in-situ hybridization, as well as in the SH-SY5Y neuroblastoma cell line differentiation model in vitro.
    RESULTS: CNTN3 gene expression was lower in cortical tubers from patients across a wide range of ages (fold change = - 0.5, p < 0.001) as compared to controls. Contactin-3 protein expression was lower in the age range of 0-3 years old (fold change = - 3.8, p < 0.001) as compared to the age-matched controls. In control brain tissue, contactin-3 gene and protein expression could be detected during fetal development, peaked around birth and during infancy and declined in the adult brain. CNTN3 expression was induced in the differentiated SH-SY5Y neuroblastoma cells in vitro (fold change = 6.2, p < 0.01).
    CONCLUSIONS: Our data show a lower expression of contactin-3 in cortical tubers of TSC patients during early postnatal period as compared to controls, which may affect normal brain development and might contribute to neuropsychiatric co-morbidities observed in patients with TSC.
    Keywords:  Cell adhesion; Cerebral cortex development; Epilepsy; Neurodevelopmental disorders; mTORopathies
    DOI:  https://doi.org/10.1186/s11689-022-09416-2
  43. Mol Cell Proteomics. 2022 Jan 07. pii: S1535-9476(22)00003-2. [Epub ahead of print] 100195
    Interactome analysis of human phospholipase D and phosphatidic acid-associated protein network.
    Rebecca Elizabeth Kattan, Han Han, Gayoung Seo, Bing Yang, Yongqi Lin, Max Dotson, Stephanie Pham, Yahya Menely, Wenqi Wang.
      Mammalian phospholipase D (PLD) enzyme family consists of six members. Among them, PLD1/2/6 catalyze phosphatidic acid (PA) production, while PLD3/4/5 have no catalytic activities. Deregulation of the PLD-PA lipid signaling has been associated with various human diseases including cancers. However, a comprehensive analysis of the regulators and effectors for this crucial lipid metabolic pathway has not been fully achieved. Using a proteomic approach, we defined the protein interaction network for the human PLD family of enzymes and PA, and revealed diverse cellular signaling events involving them. Through it, we identified PJA2 as a novel E3 ubiquitin ligase for PLD1 involved in control of the PLD1-mediated mTOR signaling. Additionally, we showed that PA interacted with and positively regulated sphingosine kinase 1 (SPHK1). Taken together, our study not only generates a rich interactome resource for further characterizing the human PLD-PA lipid signaling, but also connects this important metabolic pathway with numerous biological processes.
    Keywords:  PA; PJA2; PLD; S1P; SPHK1
    DOI:  https://doi.org/10.1016/j.mcpro.2022.100195
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