title: Multiple protective activities of neuroglobin in cultured neuronal cells exposed to hypoxia re-oxygenation injury.
abstract: Oxidative stress is associated with the pathology of acute and chronic neurodegenerative disease. We have cloned a human neuroglobin (Nb) construct and over-expressed this protein in cultured human neuronal cells to assess whether Nb ameliorates the cellular response to experimental hypoxia-reoxygenation (H/R) injury. Parental cells transfected with a blank (pDEST40) vector responded to H/R injury with a significant decrease in cellular ATP at 5 and 24 h after insult. This was coupled with increases in the cytosolic Ca(2+), and the transition metals iron (Fe), copper (Cu), and zinc (Zn) within the cell body, as monitored simultaneously using X-ray fluorescence microprobe imaging. Parental cell viability decreased over the same time period with a approximately 4 to 5-fold increase in cell death (maximum approximately 25%) matched by an increase in caspase 3/7 activation (peaking at a 15-fold increase after 24 h) and condensation of beta-actin along axonal processes. Over-expression of Nb inhibited ATP loss and except for significant decreases in the sulfur (S), chlorine (Cl), potassium (K) and Ca(2+) contents, maintained cellular ion homeostasis after H/R insult. This resulted in increased cell viability, significantly diminished caspase activation and maintenance of the beta-actin cytoskeletal structure and receptor-mediated endocytosis. These data indicate that bolstering the cellular content of Nb inhibits neuronal cell dysfunction promoted by H/R insult through multiple protective actions including: (i) maintenance of cellular bioenergetics; (ii) inhibition of Ca(2+) influx; (iii) a reduction in cellular uptake of Fe, Cu and Zn at the expense of S, Cl and K; and (iv) an enhancement of cell viability through inhibiting necrosis and apoptosis.
authors.affiliation: ANZAC Research Institute, Concord Hospital, New South Wales, Australia.
authors.forename: Thi Thuy Hong
authors.initials: TT
authors.lastname: Duong
authors.forename: Paul Kenneth
authors.initials: PK
authors.lastname: Witting
authors.forename: Shane Tony
authors.initials: ST
authors.lastname: Antao
authors.forename: Sarah Nicole
authors.initials: SN
authors.lastname: Parry
authors.forename: Marina
authors.initials: M
authors.lastname: Kennerson
authors.forename: Barry
authors.initials: B
authors.lastname: Lai
authors.forename: Stefan
authors.initials: S
authors.lastname: Vogt
authors.forename: Peter Andrew
authors.initials: PA
authors.lastname: Lay
authors.forename: Hugh Hamlyn
authors.initials: HH
authors.lastname: Harris
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000704:Analysis of Variance; D002118:Calcium / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002454:Cell Differentiation; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D003300:Copper / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004705:Endocytosis; D005434:Flow Cytometry / Q000379:methods; D005914:Globins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007501:Iron / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009447:Neuroblastoma; D000078787:Neuroglobin; D009474:Neurons / Q000187:drug effects* / Q000502:physiology*; D010100:Oxygen / Q000494:pharmacology*; D013052:Spectrometry, X-Ray Emission / Q000379:methods; D013997:Time Factors; D014162:Transfection / Q000379:methods; D015032:Zinc / Q000378:metabolism
chemical_list: D003598:Cytoskeletal Proteins; D009419:Nerve Tissue Proteins; D000078787:Neuroglobin; D003300:Copper; D000255:Adenosine Triphosphate; D005914:Globins; D007501:Iron; D020169:Caspases; D015032:Zinc; D010100:Oxygen; D002118:Calcium