Hypoxia-inducible factor (HIF) plays an important role in cell survival by regulating iron, antioxidant defense, and mitochondrial function. Pharmacological inhibitors of the iron-dependent enzyme class prolyl hydroxylases (PHD), which target α subunits of HIF proteins for degradation, have recently been demonstrated to alleviate neurodegeneration associated with stroke and hypoxic-ischemic injuries. Here we report that inhibition of PHD by 3,4-dihydroxybenzoate (DHB) protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigral dopaminergic cell loss and up-regulates HIF-1α within these neurons. Elevations in mRNA and protein levels of HIF-dependent genes heme oxygenase-1 (
Ho-1) and manganese superoxide dismutase (
Mnsod) following DHB pretreatment alone are also maintained in the presence of MPTP. MPTP-induced reductions in ferroportin and elevations in nigral and striatal iron levels were reverted to levels comparable with that of untreated controls with DHB pretreatment. Reductions in pyruvate dehydrogenase mRNA and activity resulting from MPTP were also found to be attenuated by DHB.
In vitro, the HIF pathway was activated in N27 cells grown at 3% oxygen treated with either PHD inhibitors or an iron chelator. Concordant with our
in vivo data, the MPP
+-elicited increase in total iron as well as decreases in cell viability were attenuated in the presence of DHB. Taken together, these data suggest that protection against MPTP neurotoxicity may be mediated by alterations in iron homeostasis and defense against oxidative stress and mitochondrial dysfunction brought about by cellular HIF-1α induction. This study provides novel data extending the possible therapeutic utility of HIF induction to a Parkinson disease model of neurodegeneration, which may prove beneficial not only in this disorder itself but also in other diseases associated with metal-induced oxidative stress.Parkinson disease (PD)
2 is a neurodegenerative disorder primarily associated with loss of dopaminergic (DAergic) neurons of the pars compacta region of the substantia nigra (SNpc). Dopaminergic neurons are particularly prone to oxidative damage due to high levels of inherent reactive oxygen species that are produced during dopamine synthesis or its breakdown by monoamine oxidases or autoxidation to quinones (
1–
3). Importantly, iron bound to neuromelanin within DAergic neurons can subsequently react with metabolically liberated hydrogen peroxide through the Fenton reaction to produce extremely toxic hydroxyl radicals. If not properly buffered, hydroxyl radicals can stimulate protein oxidation and lipid peroxidation, which is thought to contribute to macromolecular injury and neuronal death. Iron is the most abundant metal in the brain and some degree of accessible reactive iron is necessary for brain viability as it serves as a cofactor in DNA, RNA, and protein synthesis and for heme and non-heme enzymes involved in both mitochondrial respiration and neurotransmitter synthesis (
4). Although iron deficiencies early in life are known to result in impairments in brain development (
5), high concentrations of iron may result in cellular toxicity (
6) in part due to its ability to catalyze the production of toxic oxygen radicals.An important family of enzymes that require iron as an essential cofactor are the prolyl 4-hydroxylases (PHDs), which serve to hydroxylate proline residues situated within hypoxia-inducible factor proteins (HIFs) (
7). Under hypoxic or iron-lacking conditions, PHDs are prevented from hydroxylating proline residues within the alpha (α) subunits of the HIF protein, preventing the ubiquitination and proteasomal degradation of the protein. Stabilization of HIFα results in its accumulation within the cytosol and translocation to the nucleus where it binds HIFβ and then to hypoxia response elements found on a variety of genes including heme oxygenase-1 (
Ho-1) and manganese superoxide dismutase (
Mnsod).Previous studies have demonstrated that deferoxamine, an iron chelator, can activate HIF-1α and prevent neuronal death in both
in vitro and
in vivo models of ischemia likely via inhibition of PHDs (
8,
9). PHD inhibitors have been demonstrated to prevent oxidative cell death and ischemic injury via HIF pathway activation (
10). More recently, it has been shown that inactivation of HIF-1α in specific cortical and striatal neurons exacerbated tissue damage in a mouse model of ischemia (
11). With increasing evidence of the protective effects of induction of HIF-dependent gene products involved in iron regulation, cell survival, and energy metabolism, PHD inhibitors have been implicated as targets for neuroprotection in the central nervous system. We demonstrate here that PHD inhibition increases induction of HIF and HIF-related genes, functionally impacts on parameters of iron homeostasis and metabolic function, and, most importantly, significantly reduces the extent of DAergic nigrostriatal injury observed in the well established murine MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) PD model.
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