C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking

Manal A. Farg, Vinod Sundaramoorthy, Jessica M. Sultana, Shu Yang, Rachel A K Atkinson, Vita Levina, Mark A. Halloran, Paul A. Gleeson, Ian P. Blair, Kai Y. Soo, Anna E. King, Julie D. Atkin

    Research output: Contribution to journalArticleResearchpeer-review

    Abstract

    Intronic expansion of a hexanucleotideGGGGCCrepeat in the chromosome9 open reading frame72 (C9ORF72) geneisthemajorcauseoffamilialamyotrophic lateral sclerosis(ALS)andfrontotemporaldementia.However,the cellular function of theC9ORF72 protein remains unknown. Here,wedemonstrate thatC9ORF72 regulates endosomal trafficking. C9ORF72 colocalized with Rab proteins implicated in autophagy and endocytic transport: Rab1, Rab5, Rab7 and Rab11 in neuronal cell lines, primary cortical neurons and human spinal cord motor neurons, consistent with previous predictionsthatC9ORF72 bearsRabguanine exchange factor activity.Consistent with this notion, C9ORF72 was present in the extracellular space and as cytoplasmic vesicles. Depletion of C9ORF72 using siRNA inhibited transport of Shiga toxin fromthe plasmamembrane to Golgi apparatus, internalizationofTrkBreceptorandalteredthe ratio ofautophagosomemarker lightchain3(LC3) II:LC3I, indicatingthat C9ORF72 regulates endocytosis and autophagy. C9ORF72 also colocalized with ubiquilin-2 and LC3-positive vesicles, and co-migrated with lysosome-stained vesicles in neuronal cell lines, providing further evidence thatC9ORF72regulates autophagy. Investigationof proteinsinteractingwithC9ORF72usingmassspectrometry identified other proteins implicated in ALS; ubiquilin-2 and heterogeneous nuclear ribonucleoproteins, hnRNPA2/B1 and hnRNPA1, and actin. Treatment of cells overexpressing C9ORF72 with proteasome inhibitors induced the formation of stress granules positive for hnRNPA1 and hnRNPA2/B1. Immunohistochemistry of C9ORF72 ALS patient motor neurons revealed increased colocalization between C9ORF72 and Rab7 and Rab11 compared with controls, suggesting possible dysregulation of trafficking in patients bearing the C9ORF72 repeat expansion. Hence, this study identifies a role for C9ORF72 in Rab-mediated cellular trafficking.

    LanguageEnglish
    Article numberddu068
    Pages3579-3595
    Number of pages17
    JournalHuman molecular genetics
    Volume23
    Issue number13
    DOIs
    Publication statusPublished - 2014

    Fingerprint

    Frontotemporal Dementia
    Motor Neuron Disease
    Reading
    Autophagy
    Motor Neurons
    Cytoplasmic Vesicles
    Heterogeneous-Nuclear Ribonucleoproteins
    Shiga Toxin
    Cell Line
    Proteins
    Proteasome Inhibitors
    Extracellular Space
    Golgi Apparatus
    Endocytosis
    Lysosomes
    Small Interfering RNA
    Actins
    Spinal Cord
    Immunohistochemistry

    Cite this

    Farg, Manal A. ; Sundaramoorthy, Vinod ; Sultana, Jessica M. ; Yang, Shu ; Atkinson, Rachel A K ; Levina, Vita ; Halloran, Mark A. ; Gleeson, Paul A. ; Blair, Ian P. ; Soo, Kai Y. ; King, Anna E. ; Atkin, Julie D. / C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking. In: Human molecular genetics. 2014 ; Vol. 23, No. 13. pp. 3579-3595.
    @article{fd54cae068524826b566d595394bf968,
    title = "C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking",
    abstract = "Intronic expansion of a hexanucleotideGGGGCCrepeat in the chromosome9 open reading frame72 (C9ORF72) geneisthemajorcauseoffamilialamyotrophic lateral sclerosis(ALS)andfrontotemporaldementia.However,the cellular function of theC9ORF72 protein remains unknown. Here,wedemonstrate thatC9ORF72 regulates endosomal trafficking. C9ORF72 colocalized with Rab proteins implicated in autophagy and endocytic transport: Rab1, Rab5, Rab7 and Rab11 in neuronal cell lines, primary cortical neurons and human spinal cord motor neurons, consistent with previous predictionsthatC9ORF72 bearsRabguanine exchange factor activity.Consistent with this notion, C9ORF72 was present in the extracellular space and as cytoplasmic vesicles. Depletion of C9ORF72 using siRNA inhibited transport of Shiga toxin fromthe plasmamembrane to Golgi apparatus, internalizationofTrkBreceptorandalteredthe ratio ofautophagosomemarker lightchain3(LC3) II:LC3I, indicatingthat C9ORF72 regulates endocytosis and autophagy. C9ORF72 also colocalized with ubiquilin-2 and LC3-positive vesicles, and co-migrated with lysosome-stained vesicles in neuronal cell lines, providing further evidence thatC9ORF72regulates autophagy. Investigationof proteinsinteractingwithC9ORF72usingmassspectrometry identified other proteins implicated in ALS; ubiquilin-2 and heterogeneous nuclear ribonucleoproteins, hnRNPA2/B1 and hnRNPA1, and actin. Treatment of cells overexpressing C9ORF72 with proteasome inhibitors induced the formation of stress granules positive for hnRNPA1 and hnRNPA2/B1. Immunohistochemistry of C9ORF72 ALS patient motor neurons revealed increased colocalization between C9ORF72 and Rab7 and Rab11 compared with controls, suggesting possible dysregulation of trafficking in patients bearing the C9ORF72 repeat expansion. Hence, this study identifies a role for C9ORF72 in Rab-mediated cellular trafficking.",
    author = "Farg, {Manal A.} and Vinod Sundaramoorthy and Sultana, {Jessica M.} and Shu Yang and Atkinson, {Rachel A K} and Vita Levina and Halloran, {Mark A.} and Gleeson, {Paul A.} and Blair, {Ian P.} and Soo, {Kai Y.} and King, {Anna E.} and Atkin, {Julie D.}",
    year = "2014",
    doi = "10.1093/hmg/ddu068",
    language = "English",
    volume = "23",
    pages = "3579--3595",
    journal = "Human molecular genetics",
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    Farg, MA, Sundaramoorthy, V, Sultana, JM, Yang, S, Atkinson, RAK, Levina, V, Halloran, MA, Gleeson, PA, Blair, IP, Soo, KY, King, AE & Atkin, JD 2014, 'C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking', Human molecular genetics, vol. 23, no. 13, ddu068, pp. 3579-3595. https://doi.org/10.1093/hmg/ddu068

    C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking. / Farg, Manal A.; Sundaramoorthy, Vinod; Sultana, Jessica M.; Yang, Shu; Atkinson, Rachel A K; Levina, Vita; Halloran, Mark A.; Gleeson, Paul A.; Blair, Ian P.; Soo, Kai Y.; King, Anna E.; Atkin, Julie D.

    In: Human molecular genetics, Vol. 23, No. 13, ddu068, 2014, p. 3579-3595.

    Research output: Contribution to journalArticleResearchpeer-review

    TY - JOUR

    T1 - C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking

    AU - Farg, Manal A.

    AU - Sundaramoorthy, Vinod

    AU - Sultana, Jessica M.

    AU - Yang, Shu

    AU - Atkinson, Rachel A K

    AU - Levina, Vita

    AU - Halloran, Mark A.

    AU - Gleeson, Paul A.

    AU - Blair, Ian P.

    AU - Soo, Kai Y.

    AU - King, Anna E.

    AU - Atkin, Julie D.

    PY - 2014

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    N2 - Intronic expansion of a hexanucleotideGGGGCCrepeat in the chromosome9 open reading frame72 (C9ORF72) geneisthemajorcauseoffamilialamyotrophic lateral sclerosis(ALS)andfrontotemporaldementia.However,the cellular function of theC9ORF72 protein remains unknown. Here,wedemonstrate thatC9ORF72 regulates endosomal trafficking. C9ORF72 colocalized with Rab proteins implicated in autophagy and endocytic transport: Rab1, Rab5, Rab7 and Rab11 in neuronal cell lines, primary cortical neurons and human spinal cord motor neurons, consistent with previous predictionsthatC9ORF72 bearsRabguanine exchange factor activity.Consistent with this notion, C9ORF72 was present in the extracellular space and as cytoplasmic vesicles. Depletion of C9ORF72 using siRNA inhibited transport of Shiga toxin fromthe plasmamembrane to Golgi apparatus, internalizationofTrkBreceptorandalteredthe ratio ofautophagosomemarker lightchain3(LC3) II:LC3I, indicatingthat C9ORF72 regulates endocytosis and autophagy. C9ORF72 also colocalized with ubiquilin-2 and LC3-positive vesicles, and co-migrated with lysosome-stained vesicles in neuronal cell lines, providing further evidence thatC9ORF72regulates autophagy. Investigationof proteinsinteractingwithC9ORF72usingmassspectrometry identified other proteins implicated in ALS; ubiquilin-2 and heterogeneous nuclear ribonucleoproteins, hnRNPA2/B1 and hnRNPA1, and actin. Treatment of cells overexpressing C9ORF72 with proteasome inhibitors induced the formation of stress granules positive for hnRNPA1 and hnRNPA2/B1. Immunohistochemistry of C9ORF72 ALS patient motor neurons revealed increased colocalization between C9ORF72 and Rab7 and Rab11 compared with controls, suggesting possible dysregulation of trafficking in patients bearing the C9ORF72 repeat expansion. Hence, this study identifies a role for C9ORF72 in Rab-mediated cellular trafficking.

    AB - Intronic expansion of a hexanucleotideGGGGCCrepeat in the chromosome9 open reading frame72 (C9ORF72) geneisthemajorcauseoffamilialamyotrophic lateral sclerosis(ALS)andfrontotemporaldementia.However,the cellular function of theC9ORF72 protein remains unknown. Here,wedemonstrate thatC9ORF72 regulates endosomal trafficking. C9ORF72 colocalized with Rab proteins implicated in autophagy and endocytic transport: Rab1, Rab5, Rab7 and Rab11 in neuronal cell lines, primary cortical neurons and human spinal cord motor neurons, consistent with previous predictionsthatC9ORF72 bearsRabguanine exchange factor activity.Consistent with this notion, C9ORF72 was present in the extracellular space and as cytoplasmic vesicles. Depletion of C9ORF72 using siRNA inhibited transport of Shiga toxin fromthe plasmamembrane to Golgi apparatus, internalizationofTrkBreceptorandalteredthe ratio ofautophagosomemarker lightchain3(LC3) II:LC3I, indicatingthat C9ORF72 regulates endocytosis and autophagy. C9ORF72 also colocalized with ubiquilin-2 and LC3-positive vesicles, and co-migrated with lysosome-stained vesicles in neuronal cell lines, providing further evidence thatC9ORF72regulates autophagy. Investigationof proteinsinteractingwithC9ORF72usingmassspectrometry identified other proteins implicated in ALS; ubiquilin-2 and heterogeneous nuclear ribonucleoproteins, hnRNPA2/B1 and hnRNPA1, and actin. Treatment of cells overexpressing C9ORF72 with proteasome inhibitors induced the formation of stress granules positive for hnRNPA1 and hnRNPA2/B1. Immunohistochemistry of C9ORF72 ALS patient motor neurons revealed increased colocalization between C9ORF72 and Rab7 and Rab11 compared with controls, suggesting possible dysregulation of trafficking in patients bearing the C9ORF72 repeat expansion. Hence, this study identifies a role for C9ORF72 in Rab-mediated cellular trafficking.

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