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AraC binds the p75NTR transmembrane domain to induce neurodegeneration in mature neurons

Vanessa Lopes-Rodrigues, Pia Boxy, Eunice Sim, Dong Ik Park, Josep Carbonell, Annika Andersson, Diana Fernández-Suárez, Anders Nykjær, View ORCID ProfileLilian Kisiswa
doi: https://doi.org/10.1101/2021.12.01.470721
Vanessa Lopes-Rodrigues
1Department of Physiology and Life Sciences Institute, National University of Singapore, Singapore 117597, Singapore.
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Pia Boxy
2Danish Research Institute of Translational Neuroscience (DANDRITE)–Nordic EMBL Partnership for Molecular Medicine, Department of Biomedicine, Aarhus University, Denmark
3The Danish National Research Foundation Center, PROMEMO, Department of Biomedicine, Aarhus University, Aarhus, Denmark
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Eunice Sim
1Department of Physiology and Life Sciences Institute, National University of Singapore, Singapore 117597, Singapore.
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Dong Ik Park
2Danish Research Institute of Translational Neuroscience (DANDRITE)–Nordic EMBL Partnership for Molecular Medicine, Department of Biomedicine, Aarhus University, Denmark
3The Danish National Research Foundation Center, PROMEMO, Department of Biomedicine, Aarhus University, Aarhus, Denmark
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Josep Carbonell
4Department of Neuroscience, Karolinska Institute, Stockholm S-17177, Sweden
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Annika Andersson
4Department of Neuroscience, Karolinska Institute, Stockholm S-17177, Sweden
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Diana Fernández-Suárez
4Department of Neuroscience, Karolinska Institute, Stockholm S-17177, Sweden
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Anders Nykjær
2Danish Research Institute of Translational Neuroscience (DANDRITE)–Nordic EMBL Partnership for Molecular Medicine, Department of Biomedicine, Aarhus University, Denmark
3The Danish National Research Foundation Center, PROMEMO, Department of Biomedicine, Aarhus University, Aarhus, Denmark
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Lilian Kisiswa
2Danish Research Institute of Translational Neuroscience (DANDRITE)–Nordic EMBL Partnership for Molecular Medicine, Department of Biomedicine, Aarhus University, Denmark
3The Danish National Research Foundation Center, PROMEMO, Department of Biomedicine, Aarhus University, Aarhus, Denmark
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  • ORCID record for Lilian Kisiswa
  • For correspondence: liki@biomed.au.dk
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Abstract

Background Cytosine arabinoside (AraC) is one of the main therapeutic treatments for several types of cancer including acute myeloid leukaemia. However, after high dose AraC chemotherapy regime, patients develop severe neurotoxicity and neurodegeneration in the central nervous system leading to cerebellar ataxia, dysarthria, nystagmus, somnolence and drowsiness. AraC induces apoptosis in dividing cells, however, the mechanism by which it leads to neurite degeneration and cell death in mature neurons remains unclear. We hypothesized that the upregulation of the death receptor p75NTR is responsible for AraC-mediated neurodegeneration and cell death in leukemia patients undergoing AraC treatment.

Methods To determine the role of AraC-p75NTR signalling in degeneration of mature cerebellar granule neurons, we used primary cultures from p75NTR knockout and p75NTRCys259 mice. Evaluation of neurodegeneration, cell death and p75NTR signalling was done by immunohistochemistry and immunoblotting. To assess the direct interaction between AraC and p75NTR, we performed isothermal dose response-cellular thermal shift and AraTM assays as well as Homo-FRET anisotropy imaging.

Results We show that AraC induces neurite degeneration and programmed cell death of mature cerebellar granule neurons in a p75NTR-dependent manner. Mechanistically, AraC binds to Proline 252 and Cysteine 256 of the p75NTR transmembrane domain and selectively uncouples p75NTR from the NFκB survival pathway. This in turn, exacerbates the activation of the cell death/JNK pathway by recruitment of TRAF6 to p75NTR.

Conclusion Our findings identify p75NTR as a novel molecular target to develop treatments to counteract AraC-mediated neurodegeneration.

Competing Interest Statement

The authors have declared no competing interest.

  • Abbreviations

    AKT
    protein kinase B
    ALL
    acute lymphatic leukaemia
    AML
    acute myeloid leukaemia
    ANOVA
    analysis of variance
    AraC
    1-beta-D-arabinofuranosyl-cytosine cytarabine;
    AraTM
    ara transmembrane;
    BCA
    bicinchoninic acid assay
    cDNA
    complementary DNA
    CETSA
    the cellular thermal shift assay,
    CETSA
    the cellular thermal shift assay,
    CGNs
    cerebellar granule neurons;
    CNS
    centran nervous system
    COS-7
    -African green monkey kidney fibroblast-like cell line.
    Cys
    cystein
    DI
    degeneration index
    DIV
    days in vitro
    DMSO
    dimethyl sufoxide
    DNA
    deoxyribonucleic acid
    ECL
    enhanced chemiluminescence
    EGFP
    enhanced green fluorescent protein
    FRET
    förster Resonance Energy Transfer;
    GAPDH
    glyceraldehyde 3-phosphate dehydrogenase
    HEK
    human embryonic kidney cells
    HEPES
    4-(2-hydroxyethyl1)-1-piperazineethanesulfonic acid
    HIDAC
    high Dose AraC
    HRP
    horseradish peroxidase
    IgG
    immunoglobulin
    IkBa
    nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha
    Ile
    isoleucin
    IPTG
    isopropyl B-D-1-thiogalactopyranoside
    ITDR-CETSA
    Isothermal dose response-cellular thermal shift assay
    JNK
    c-Jun N-terminal kinase
    KCL
    potassium chloride
    LB
    lysogeny broth
    NFkB
    nuclear factor kappa-light-chain-enhancer of activated B cells
    NIH
    national institute of health
    NTR
    neurotrophine receptor
    PBS
    phosphate buffered solution
    PCR
    polymeras chain reaction
    PLA
    Proximity Ligation Assay
    RhoA
    ras homolog family member A
    RhoGDI
    Rho GDP-dissociation inhibitor
    RT
    room temperature
    Ser
    serin
    TAG-1
    transiet axonal glycoprotein
    TMD
    transmembrane domain
    TNF
    trumor necrosis factor
    TRAF6
    TNF receptor associated factor 6
    TrkB
    Tropomysin receptor kinase B
    TUNEL
    terminal deoxynucleotidyl transferase dUTP nick end labelling
    Val
    valin
    WT
    wild type
  • Copyright 
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    AraC binds the p75NTR transmembrane domain to induce neurodegeneration in mature neurons
    Vanessa Lopes-Rodrigues, Pia Boxy, Eunice Sim, Dong Ik Park, Josep Carbonell, Annika Andersson, Diana Fernández-Suárez, Anders Nykjær, Lilian Kisiswa
    bioRxiv 2021.12.01.470721; doi: https://doi.org/10.1101/2021.12.01.470721
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    AraC binds the p75NTR transmembrane domain to induce neurodegeneration in mature neurons
    Vanessa Lopes-Rodrigues, Pia Boxy, Eunice Sim, Dong Ik Park, Josep Carbonell, Annika Andersson, Diana Fernández-Suárez, Anders Nykjær, Lilian Kisiswa
    bioRxiv 2021.12.01.470721; doi: https://doi.org/10.1101/2021.12.01.470721

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