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