Multiple protective activities of neuroglobin in cultured neuronal cells exposed to hypoxia re-oxygenation injury.
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.
Duong
Thi Thuy Hong
TT
ANZAC Research Institute, Concord Hospital, New South Wales, Australia.
Witting
Paul Kenneth
PK
Antao
Shane Tony
ST
Parry
Sarah Nicole
SN
Kennerson
Marina
M
Lai
Barry
B
Vogt
Stefan
S
Lay
Peter Andrew
PA
Harris
Hugh Hamlyn
HH
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
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