Abstract
Neurotrophins are critical for the survival of neurons during development and insufficient access to neurotrophins later in life may contribute to the loss of neurons in neurodegenerative disease, spinal cord injury, and stroke. The prolyl hydroxylase inhibitors ethyl 3,4-dihydroxybenzoic acid (DHB) and dimethyloxalylglycine (DMOG) were shown to inhibit cell death in a model of neurotrophin deprivation that involves depriving sympathetic neurons of nerve growth factor (NGF). Here we show that treatment with DMOG or DHB reverses the decline in 2-deoxyglucose uptake caused by NGF withdrawal and suppresses the NGF deprivation-induced accumulation of reactive oxygen species. Neither DMOG nor DHB prevented death when NGF deprivation was carried out under conditions of glucose starvation, and both compounds proved toxic to NGF-maintained neurons deprived of glucose, suggesting that their survival-promoting effects are mediated through the preservation of glucose metabolism. DHB and DMOG are well known activators of hypoxia-inducible factor (HIF), but whether activation of HIF underlies their survival-promoting effects is not known. Using gene disruption and RNA interference, we provide evidence that DMOG and, to a lesser extent, DHB require HIF-2α expression to inhibit NGF deprivation-induced death. Furthermore, suppressing basal HIF-2α expression, but not HIF-1α, in NGF-maintained neurons is sufficient to promote cell death. These results implicate HIF-2α in the neuroprotective mechanisms of prolyl hydroxylase inhibitors and in an endogenous cell survival pathway activated by NGF in developing neurons.
Footnotes
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This work was supported by the National Institutes of Health National Institute of Neurological Disorders and Stroke [Grants NS34400, NS58868, NS37110].
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ABBREVIATIONS: SCG, superior cervical ganglion; CM-H2DCFDA, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; cpt-cAMP, 8-(4-chlorophenylthio)-cAMP; DHB, ethyl 3,4-dihydroxybenzoic acid; DMOG, dimethyloxalylglycine; GFP, green fluorescent protein; 4-HT, 4-hydroxytamoxifen; HIF, hypoxia-inducible factor; JNK, c-Jun N-terminal kinase; NGF, nerve growth factor; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; ROS, reactive oxygen species; TBS, Tris-buffered saline; HIF, hypoxia-inducible factor; JNK, c-Jun NH2-terminal kinase; 4-HT, 4-hydroxytamoxifen; shRNA, short hairpin RNA; GFP, green fluorescent protein; 2DOG, [3H]2-deoxy-d-glucose; ANOVA, analysis of variance.
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↵1 Current affiliation: Department of Pathology, Harvard Medical School, Boston, Massachusetts.
- Received October 30, 2008.
- Accepted February 9, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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