Abstract
Hypoxia-inducible factor-1 (HIF-1) plays an important role in neural progenitor cell (NPC) propagation and dopaminergic differentiation. In the presence of oxygen and iron, hypoxia-inducible factor 1 alpha (HIF-1α) is rapidly degraded via the prolyl hydroxylase (PHD)/VHL pathway. In addition to hypoxia, various non-hypoxic stimuli can stabilize HIF-1α in NPCs and influence the transcription of HIF-regulated genes. Here, we investigate various hypoxia mimetics: deferoxamine (DFO), ciclopirox olamine (CPX), dimethyloxallyl glycine (DMOG), a novel HIF-PHD inhibitor (FG-4497) and cobalt chloride (CoCl2) with respect to their ability to enhance in vitro proliferation, neurogenesis and dopaminergic differentiation of human fetal mesencephalic NPCs (hmNPCs) in ambient oxygen (21%). Although able to stabilize HIF-1α, iron chelators (DFO and CPX) and DMOG were toxic to hmNPCs. CoCl2 was beneficial only towards neuronal and dopaminergic differentiation, while FG-4497 enhanced proliferation, neurogenesis and dopaminergic differentiation of hmNPCs. Both CoCl2 and FG-4497 were protective to human dopaminergic neurons. Finally, exposure to hyperbaric oxygen (HBO) also stabilized HIF-1α in hmNPCs and induced neurogenesis in vitro. These findings suggest that several HIF stabilizing agents or conditions can rescue impaired neurons and promote neurogenesis in vitro.
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Abbreviations
- ANOVA:
-
Analysis of variance
- CPX:
-
Ciclopirox olamine
- DA:
-
Dopamine
- DAPI:
-
6′-Diamidino-2-phenylindole
- DAT:
-
Dopamine transporter
- DCX:
-
Doublecortin
- DFO:
-
Deferoxamine mesylate
- DMOG:
-
Dimethyloxallyl glycine
- EGF:
-
Epidermal growth factor
- EPO:
-
Erythropoietin
- FCS:
-
Fetal calf serum
- FGF-2:
-
Fibroblast growth factor 2
- GFAP:
-
Glial fibrillary acidic protein
- HBO:
-
Hyperbaric oxygen
- HEK:
-
Human embryonic kidney
- HIF-1α:
-
Hypoxia-inducible factor-1α
- hmNPCs:
-
Human mesencephalic neural progenitor cells
- hfNPCs:
-
Human frontal (cortical) neural progenitor cells
- MAP2ab:
-
Microtubule associated protein-2
- MPP+ :
-
1-Methyl-4-phenylpyridinium
- MTT:
-
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
- NSE:
-
Neuron-specific enolase
- PHD:
-
Prolyl 4-hydroxylase domain
- PHI:
-
PHD inhibitor
- TH:
-
Tyrosine hydroxylase
- TUJ1:
-
Neuronal class III β-tubulin
- VEGF:
-
Vascular endothelial growth factor
- pVHL:
-
von Hippel–Lindau tumour suppressor protein
References
Bruick RK, McKnight SL (2001) A conserved family of prolyl-4-hydroxylases that modify HIF. Science 294:1337–1340
Brunelle JK, Bell EL, Quesada NM, Vercauteren K, Tiranti V, Zeviani M, Scarpulla RC, Chandel NS (2005) Oxygen sensing requires mitochondrial ROS but not oxidative phosphorylation. Cell Metab 1:409–414
Chachami G, Simos G, Hatziefthimiou A, Bonanou S, Molyvdas PA, Paraskeva E (2004) Cobalt induces hypoxia-inducible factor-1alpha expression in airway smooth muscle cells by a reactive oxygen species- and PI3K-dependent mechanism. Am J Respir Cell Mol Biol 31:544–551
Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, Schumacker PT (1998) Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci USA 95:11715–11720
Chen HL, Pistollato F, Hoeppner DJ, Ni HT, McKay RDG, Panchision DM (2007a) Oxygen tension regulates survival and fate of mouse central nervous system precursors at multiple levels. Stem Cells 25:2291–2301
Chen ZY, Asavaritikrai P, Prchal JT, Noguchi CT (2007b) Endogenous erythropoietin signaling is required for normal neural progenitor cell proliferation. J Biol Chem 282:25875–25883
Csete M, Rodriguez L, Wilcox M, Chadalavada S (2004) Erythropoietin receptor is expressed on adult rat dopaminergic neurons and erythropoietin is neurotrophic in cultured dopaminergic neuroblasts. Neurosci Lett 359:124–126
Danet GH, Pan Y, Luongo JL, Bonnet DA, Simon MC (2003) Expansion of human SCID-repopulating cells under hypoxic conditions. J Clin Invest 112:126–135
Demers EJ, McPherson RJ, Juul SE (2005) Erythropoietin protects dopaminergic neurons and improves neurobehavioral outcomes in juvenile rats after neonatal hypoxia-ischemia. Pediatr Res 58:297–301
Goldberg M, Dunning S, Bunn H (1988) Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science 242:1412–1415
Grayson WL, Zhao F, Izadpanah R, Bunnell B, Ma T (2006) Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs. J Cell Physiol 207:331–339
Guzy RD, Schumacker PT (2006) Oxygen sensing by mitochondria at complex III: the paradox of increased reactive oxygen species during hypoxia. Exp Physiol 91:807–819
Hsieh MM, Linde NS, Wynter A, Metzger M, Wong C, Langsetmo I, Lin A, Smith R, Rodgers GP, Donahue RE, Klaus SJ, Tisdale JF (2007) HIF prolyl hydroxylase inhibition results in endogenous erythropoietin induction, erythrocytosis, and modest fetal hemoglobin expression in rhesus macaques. Blood 110:2140–2147
Hu X, Yu SP, Fraser JL, Lu Z, Ogle ME, Wang J-A, Wei L (2008) Transplantation of hypoxia-preconditioned mesenchymal stem cells improves infarcted heart function via enhanced survival of implanted cells and angiogenesis. J Thorac Cardiovasc Surg 135:799–808
Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, Salic A, Asara JM, Lane WS, Kaelin WG (2001) HIF alpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 292:464–468
Ivanovic Z, Dello Sbarba P, Trimoreau F, Faucher JL, Praloran V (2000) Primitive human HPCs are better maintained and expanded in vitro at 1 percent oxygen than at 20 percent. Transfusion 40:1482–1488
Jaakkola P (2001) Targeting of HIF-alpha to the von Hippel–Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292:468–472
Jin KL, Mao XO, Greenberg DA (2000) Vascular endothelial growth factor: direct neuroprotective effect in in vitro ischemia. Proc Natl Acad Sci USA 97:10242–10247
Jin K, Zhu Y, Sun Y, Mao XO, Xie L, Greenberg DA (2002) Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo. Proc Natl Acad Sci USA 99:11946–11950
Jung TW, Lee JY, Shim WS, Kang ES, Kim SK, Ahn CW, Lee HC, Cha BS (2007) Rosiglitazone protects human neuroblastoma SH-SY5Y cells against MPP+ induced cytotoxicity via inhibition of mitochondrial dysfunction and ROS production. J Neurol Sci 253:53–60
Kilic E, Kilic U, Soliz J, Bassetti CL, Gassmann M, Hermann DM (2005) Brain-derived erythropoietin protects from focal cerebral ischemia by dual activation of ERK-1/-2 and Akt pathways. FASEB J 19:2026–2028
Kim TS, Misumi S, Jung CG, Masuda T, Isobe Y, Furuyama F, Nishino H, Hida H (2008) Increase in dopaminergic neurons from mouse embryonic stem cell-derived neural progenitor/stem cells is mediated by hypoxia inducible factor-1alpha. J Neurosci Res 86:2353–2362
Kotake-Nara E, Takizawa S, Quan J, Wang H, Saida K (2005) Cobalt chloride induces neurite outgrowth in rat pheochromocytoma PC-12 cells through regulation of endothelin-2/vasoactive intestinal contractor. J Neurosci Res 81:563–571
Liu J, Narasimhan P, Yu F, Chan PH (2005) Neuroprotection by hypoxic preconditioning involves oxidative stress-mediated expression of hypoxia-inducible factor and erythropoietin. Stroke 36:1264–1269
Lopez-Lazaro M (2006) HIF-1: hypoxia-inducible factor or dysoxia-inducible factor? FASEB J 20:828–832
Lovejoy DB, Richardson DR (2002) Novel “hybrid” iron chelators derived from aroylhydrazones and thiosemicarbazones demonstrate selective antiproliferative activity against tumor cells. Blood 100:666–676
Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME, Wykoff CC, Pugh CW, Maher ER, Ratcliffe PJ (1999) The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399:271–275
Milosevic J, Schwarz SC, Krohn K, Poppe M, Storch A, Schwarz J (2005) Low atmospheric oxygen avoids maturation, senescence and cell death of murine mesencephalic neural precursors. J Neurochem 92:718–729
Milosevic J, Brandt A, Roemuss U, Arnold A, Wegner F, Schwarz SC, Storch A, Zimmermann H, Schwarz J (2006) Uracil nucleotides stimulate human neural precursor cell proliferation and dopaminergic differentiation: involvement of MEK/ERK signalling. J Neurochem 99:913–923
Milosevic J, Maisel M, Wegner F, Leuchtenberger J, Wenger RH, Gerlach M, Storch A, Schwarz J (2007a) Lack of hypoxia-inducible factor-1 alpha impairs midbrain neural precursor cells involving vascular endothelial growth factor signaling. J Neurosci 27:412–421
Milosevic J, Schwarz SC, Maisel M, Poppe-Wagner M, Dieterlen MT, Storch A, Schwarz J (2007b) Dopamine D2/D3 receptor stimulation fails to promote dopaminergic neurogenesis of murine and human midbrain-derived neural precursor cells in vitro. Stem Cells Dev 16:625–635
Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63
Nicoletti I, Migliorati G, Pagliacci M, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279
Pacary E, Legros H, Valable S, Duchatelle P, Lecocq M, Petit E, Nicole O, Bernaudin M (2006) Synergistic effects of CoCl2 and ROCK inhibition on mesenchymal stem cell differentiation into neuron-like cells. J Cell Sci 119:2667–2678
Pacary E, Tixier E, Coulet F, Roussel S, Petit E, Bernaudin M (2007) Crosstalk between HIF-1 and ROCK pathways in neuronal differentiation of mesenchymal stem cells, neurospheres and in PC12 neurite outgrowth. Mol Cell Neurosci 35:409–423
Parmar K, Mauch P, Vergilio JA, Sackstein R, Down JD (2007) Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia. Proc Natl Acad Sci USA 104:5431–5436
Pasha Z, Wang Y, Sheikh R, Zhang D, Zhao T, Ashraf M (2008) Preconditioning enhances cell survival and differentiation of stem cells during transplantation in infarcted myocardium. Cardiovasc Res 77:134–142
Prass K, Scharff A, Ruscher K, Lowl D, Muselmann C, Victorov I, Kapinya K, Dirnagl U, Meisel A (2003) Hypoxia-induced stroke tolerance in the mouse is mediated by erythropoietin. Stroke 34:1981–1986
Robinson A, Keely S, Karhausen J, Gerich ME, Furuta GT, Colgan SP (2008) Mucosal protection by hypoxia-inducible factor prolyl hydroxylase inhibition. Gastroenterology 134:145–155
Rosenberger C, Rosen S, Shina A, Frei U, Eckardt KU, Flippin LA, Arend M, Klaus SJ, Heyman SN (2008) Activation of hypoxia inducible factors (HIF) ameliorates hypoxic distal tubular injury in the isolated perfused rat kidney. Nephrol Dial Transplant 23:3472–3478
Sabolek M, Mieskes I, Lenk T, Lehmensiek V, Hermann A, Schwarz J, Storch A (2008) Stage-dependent vulnerability of fetal mesencephalic neuroprogenitors towards dopaminergic neurotoxins. Neurotoxicology 29:714–721
Sanders SP, Zweier JL, Kuppusamy P, Harrison SJ, Bassett DJ, Gabrielson EW, Sylvester JT (1993) Hyperoxic sheep pulmonary microvascular endothelial cells generate free radicals via mitochondrial electron transport. J Clin Invest 91:46–52
Schaenzer A, Wachs F, Wilhelm D, Acker T, Cooper-Kuhn C, Beck H, Winkler J, Aigner L, Plate K, Kuhn HG (2004) Direct stimulation of adult neural stem cells in vitro and neurogenesis in vivo by vascular endothelial growth factor. Brain Pathol 14:237–248
Schwarz J, Schwarz SC, Storch A (2006) Developmental perspectives on human midbrain-derived neural stem cells. Neurodegener Dis 3:45–49
Semenza GL (2000) HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J Appl Physiol 88:1474–1480
Siddiq A, Ayoub IA, Chavez JC, Aminova L, Shah S, LaManna JC, Patton SM, Connor JR, Cherny RA, Volitakis I, Bush AI, Langsetmo I, Seeley T, Gunzler V, Ratan RR (2005) Hypoxia-inducible factor prolyl 4-hydroxylase inhibition: a target for neuroprotection in the central nervous system. J Biol Chem 280:41732–41743
Storch A, Ludolph AC, Schwarz J (1999) HEK-293 cells expressing the human dopamine transporter are susceptible to low concentrations of 1-methyl-4-phenylpyridine (MPP+) via impairment of energy metabolism. Neurochem Int 35:393–403
Storch A, Paul G, Csete M, Boehm BO, Carvey PM, Kupsch A, Schwarz J (2001) Long-term proliferation and dopaminergic differentiation of human mesencephalic neural precursor cells. Exp Neurol 170:317–325
Studer L, Csete M, Lee SH, Kabbani N, Walikonis J, Wold B, McKay R (2000) Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen. J Neurosci 20:7377–7383
Theus MH, Wei L, Cui L, Francis K, Hu X, Keogh C, Yu SP (2008) In vitro hypoxic preconditioning of embryonic stem cells as a strategy of promoting cell survival and functional benefits after transplantation into the ischemic rat brain. Exp Neurol 210:656–670
Tsai PT, Ohab JJ, Kertesz N, Groszer M, Matter C, Gao J, Liu X, Wu H, Carmichael ST (2006) A critical role of erythropoietin receptor in neurogenesis and post-stroke recovery. J Neurosci 26:1269–1274
Wenger RH, Stiehl DP, G Camenisch (2005) Integration of oxygen signaling at the consensus HRE. Sci STKE 2005(306):re12
Won YJ, Yoo JY, Lee JH, Hwang SJ, Kim D, Hong HN (2007) Erythropoietin is neuroprotective on GABAergic neurons against kainic acid-excitotoxicity in the rat spinal cell cultures. Brain Res 1154:31–39
Yasuhara T, Shingo T, Muraoka K, Kameda M, Agari T, Wen Ji Y, Hayase H, Hamada H, Borlongan CV, Date I (2005) Neurorescue effects of VEGF on a rat model of Parkinson’s disease. Brain Res 1053:10–18
Yuan Y, Hilliard G, Ferguson T, Millhorn DE (2003) Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel–Lindau protein by direct binding to hypoxia-inducible factor-alpha. J Biol Chem 278:15911–15916
Zhu LL, Wu LY, Yew DT, Fan M (2005) Effects of hypoxia on the proliferation and differentiation of NSCs. Mol Neurobiol 31:231–242
Acknowledgments
This work was supported by the German Federal Ministry of Education and Research (BMBF, PtJ-Bio 0313909) and the IZKF-Leipzig (TP C27).
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Milosevic, J., Adler, I., Manaenko, A. et al. Non-hypoxic Stabilization of Hypoxia-Inducible Factor Alpha (HIF-α): Relevance in Neural Progenitor/Stem Cells. Neurotox Res 15, 367–380 (2009). https://doi.org/10.1007/s12640-009-9043-z
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DOI: https://doi.org/10.1007/s12640-009-9043-z