Abstract
Cells can adapt to hypoxia through the activation of hypoxia-inducible factor-1 (HIF-1), which in turn regulates the expression of hypoxia-responsive genes. Defects in hypoxic signaling have been suggested to underlie the degeneration of motoneurons in amyotrophic lateral sclerosis (ALS). We have recently identified mutations in the hypoxia-responsive gene, angiogenin (ANG), in ALS patients, and have shown that ANG is constitutively expressed in motoneurons. Here, we show that HIF-1α is sufficient and required to activate ANG in cultured motoneurons exposed to hypoxia, although ANG expression does not change in a transgenic ALS mouse model or in sporadic ALS patients. Administration of recombinant ANG or expression of wild-type ANG protected motoneurons against hypoxic injury, whereas gene silencing of ang1 significantly increased hypoxia-induced cell death. The previously reported ALS-associated ANG mutations (Q12L, K17I, R31K, C39W, K40I, I46V) all showed a reduced neuroprotective activity against hypoxic injury. Our data show that ANG plays an important role in endogenous protective pathways of motoneurons exposed to hypoxia, and suggest that loss of function rather than loss of expression of ANG is associated with ALS.
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Abbreviations
- ALS:
-
amyotrophic lateral sclerosis
- ANG:
-
angiogenin
- ARE:
-
adenylate/uridylate-rich element
- BSA:
-
bovine serum albumin
- CSF:
-
cerebrospinal fluid
- DAPI:
-
4′,6 diamidino-2-phenylindole
- DFO:
-
deferoxamine
- DMEM:
-
Dulbecco's modified Eagle's medium
- ECL:
-
enhanced chemiluminescence
- FBS:
-
fetal bovine serum
- flk-1/vegfr2 :
-
vascular endothelial growth factor receptor 2
- HIF-1:
-
hypoxia-inducible factor-1
- HRE:
-
hypoxic response element
- HRP:
-
horseradish peroxidase
- MAP-2:
-
microtubule-associated protein 2
- MTT:
-
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- NeuN:
-
neuronal nuclei
- PBS:
-
phosphate-buffered saline
- SMI-32:
-
200 kDa neurofilament heavy antibody
- SOD1 :
-
superoxide dismutase-1
- TBS:
-
Tris-buffered saline
- VEGF:
-
vascular endothelial growth factor
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Acknowledgements
NSC34 cells (Dr. N Cashman, University of Toronto, Canada) were obtained from Professor PJ Shaw (University of Sheffield, UK). We thank the MRC UK Brain Bank (Kings College London, UK) for post-mortem spinal cord tissue from ALS and non-ALS patients. Angiogenin promoter constructs were a kind gift from Dr. Kimberly D Dyer (NIH, Bethesda, MD, USA). The HIF-1 overexpression plasmids were a kind gift from Dr. Thilo Hagen (National University, Singapore). This work was supported by funding from Science Foundation Ireland to JHMP and CTT (03/RP1/B344 and 06/UR/B920) and from Enterprise Ireland to DK (PC 2007/045).
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Sebastià, J., Kieran, D., Breen, B. et al. Angiogenin protects motoneurons against hypoxic injury. Cell Death Differ 16, 1238–1247 (2009). https://doi.org/10.1038/cdd.2009.52
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DOI: https://doi.org/10.1038/cdd.2009.52
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