Inhibition of Prolyl Hydroxylase Protects against 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Neurotoxicity

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. 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) 2The abbreviations used are: PDParkinson diseaseHIFhypoxia inducible factorDAdopamineMPTP1-methyl-4-phenyl-1,2,3,6-tetrahydropyridineDMOGdimethyloxaloylglycineDHBdihydroxybenzoateSIHsalicylaldehyde isonicotinoyl hydrazonePHDprolyl 4-hydroxylaseHO-1heme oxygenase-1SNsubstantia nigraSTstriatumVEGFvascular endothelial growth factorMnSODmanganese superoxide dismutaseTHtyrosine hydroxylaseMPTP1-methyl-4-phenyl-1,2,3,6-tetrahydropyridineCQclioquinolHPLChigh pressure liquid chromatographyRTreverse transcriptaseBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolTES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acidFPTferroportinICP-MSinductively coupled plasma-mass spectrometryPDHpyruvate dehydrogenaseSNpcsubstantia nigra pars compacta. 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 (1Haavik J. Almås B. Flatmark T. J. Neurochem. 1997; 68: 328-332Crossref PubMed Scopus (76) Google Scholar, 2Cohen G. Farooqui R. Kesler N. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 4890-4894Crossref PubMed Scopus (249) Google Scholar, 3Graham D.G. Tiffany S.M. Bell Jr., W.R. Gutknecht W.F. Mol. Pharmacol. 1978; 14: 644-653PubMed Google Scholar). 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 (4Ward R.J. Dexter D. Florence A. Aouad F. Hider R. Jenner P. Crichton R.R. Biochem. Pharmacol. 1995; 49: 1821-1826Crossref PubMed Scopus (58) Google Scholar). Although iron deficiencies early in life are known to result in impairments in brain development (5Connor J.R. Menzies S.L. J. Neurol. Sci. 1995; 134: 33-44Abstract Full Text PDF PubMed Scopus (182) Google Scholar), high concentrations of iron may result in cellular toxicity (6Tanaka M. Sotomatsu A. Kanai H. Hirai S. J. Neurol. Sci. 1991; 101: 198-203Abstract Full Text PDF PubMed Scopus (137) Google Scholar) in part due to its ability to catalyze the production of toxic oxygen radicals. Parkinson disease hypoxia inducible factor dopamine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine dimethyloxaloylglycine dihydroxybenzoate salicylaldehyde isonicotinoyl hydrazone prolyl 4-hydroxylase heme oxygenase-1 substantia nigra striatum vascular endothelial growth factor manganese superoxide dismutase tyrosine hydroxylase 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine clioquinol high pressure liquid chromatography reverse transcriptase 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid ferroportin inductively coupled plasma-mass spectrometry pyruvate dehydrogenase substantia nigra pars compacta. 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) (7Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). 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 (8Hurn P.D. Koehler R.C. Blizzard K.K. Traystman R.J. Stroke. 1995; 26 (discussion 694–685): 688-694Crossref PubMed Google Scholar, 9Hamrick S.E. McQuillen P.S. Jiang X. Mu D. Madan A. Ferriero D.M. Neurosci. Lett. 2005; 379: 96-100Crossref PubMed Scopus (102) Google Scholar). PHD inhibitors have been demonstrated to prevent oxidative cell death and ischemic injury via HIF pathway activation (10Siddiq A. Ayoub I.A. Chavez J.C. Aminova L. Shah S. LaManna J.C. Patton S.M. Connor J.R. Cherny R.A. Volitakis I. Bush A.I. Langsetmo I. Seeley T. Gunzler V. Ratan R.R. J. Biol. Chem. 2005; 280: 41732-41743Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). 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 (11Baranova O. Miranda L.F. Pichiule P. Dragatsis I. Johnson R.S. Chavez J.C. J. Neurosci. 2007; 27: 6320-6332Crossref PubMed Scopus (301) Google Scholar). With increasing evidence of the protective of induction of HIF-dependent involved in iron cell and PHD inhibitors have been as for in the that PHD inhibition induction of HIF and on of iron homeostasis and most the of DAergic injury in the well MPTP used in this study were to on a and maintained in a in the were by and to on the of in 3,4-dihydroxybenzoate (DHB) studies 3,4-dihydroxybenzoate was to a of and to to of either or of MPTP clioquinol studies of were by with either or for to the MPTP studies by the were maintained on a by L. S. J. Neurosci. 2007; 27: PubMed Scopus Google controls of were also treated with either MPTP or following the MPTP or in the were for either tissue for or brain via for of were to with by were then in and at was against tyrosine hydroxylase by and development to DAergic neurons the cells were the D. F. S. J. R. V. R. L. D. I. L. Cherny R.A. Bush A.I. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). with were to were and in acid were for of dopamine and its and acid by as in our W.R. J. Neurochem. PubMed Scopus Google Scholar, W.R. P. T. J. Neurol. Sci. Full Text PDF PubMed Scopus Google Scholar). of was through an with a and and by a of and acid are as of protein. protein concentrations of tissue were to the protein and the tissue from was for and of the and reverse for of were were as to be used on reaction for the genes were against the the and genes that the were as the for the protein and the of was by a the and reverse from the for the reaction at for at for and at for for and for was then both the as well as the genes to the for the substantia nigra were and in a of inhibitors and and with the and protein concentrations were the protein were on to were with in with HIF-1α and vascular endothelial growth factor was used as a were via for and resulting were and by of of the from were and for were and in a for of with for specific ferroportin were to the for at were used for of of dopaminergic striatal were with by and were on a of from were used for iron were for in in acid at and subsequently in of and for and were to of the to brain were to and for in of to and in for Pharmacol. PubMed Scopus Google Scholar). iron was a tissue was in and by in a were at for at was and the in and of the was and the were was at for at the was in of for pyruvate dehydrogenase enzyme activity the enzyme was within the of the and the of to was at N27 cells were at 3% to oxygen and treated with PHD inhibitors and dimethyloxaloylglycine or the iron isonicotinoyl hydrazone in the presence and of a of were with and for to translocation of iron of cells were with for treated with PHD inhibitors and then for viability was by cells with a toxic of in the presence or of DHB and was via the the of is of activity were by for data, with on the that an inhibition of iron-dependent PHD activity result in the accumulation of HIF-1α and a activation of it was that DHB may the of DAergic injury by MPTP in DHB is a of PHD that or for PHD activity Günzler V. R. Kivirikko K.I. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and has been shown to by (10Siddiq A. Ayoub I.A. Chavez J.C. Aminova L. Shah S. LaManna J.C. Patton S.M. Connor J.R. Cherny R.A. Volitakis I. Bush A.I. Langsetmo I. Seeley T. Gunzler V. Ratan R.R. J. Biol. 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PubMed Scopus Google Scholar). our this study is the to in both in vitro and in vivo the beneficial of PHD inhibition in a neurodegenerative model of a novel therapeutic pathway in of this of these studies are in of HIFα resulting from PHD inhibition can to the of proteins involved in iron synthesis and mitochondrial and the associated with MPTP. studies are necessary to the involved in the protective of PHD inhibition as well the of HIF-1α and in protection in the MPTP and for on the