Neuroglobin-overexpression alters hypoxic response gene expression in primary neuron culture following oxygen glucose deprivation

doi:10.1016/j.neuroscience.2009.04.055

Neuroscience

Volume 162, Issue 2, 18 August 2009, Pages 396-403

https://doi.org/10.1016/j.neuroscience.2009.04.055 Get rights and content

Abstract

Neuroglobin (Ngb) is a tissue globin specifically expressed in neurons. Our laboratory and others have shown that Ngb overexpression protects neurons against hypoxia/ischemia, but the underlying mechanisms remain poorly understood. Recent studies demonstrate that hypoxia/ischemia induces a multitude of spatially and temporally regulated responses in gene expression, and initial evidence suggested that Ngb might function in altering biological processes of gene expression. In this study, we asked how Ngb may help regulate genes responsive to hypoxia. Expression of hypoxic response genes following oxygen–glucose deprivation (OGD) was examined using mRNA arrays in neuroglobin-overexpressing transgenic (Ngb-Tg) and wild type (WT) mouse neurons. From a total of 113 genes on the microarray, mRNA expression of 65 genes was detected. Under rest condition, 14 genes were downregulated in Ngb-Tg neurons compared to WT. In WT neurons, after 4-h OGD followed by 4-h reoxygenation (O4/R4), 20 genes were significantly downregulated, and only Fos mRNA was significantly increased. However, out of the 20 downregulated genes in WT neurons, 12 of them were no longer significantly changed in Ngb-Tg neurons: Add1, Arnt2, Camk2g, Cstb, Dr1, Epas1, Gna11, Hif1a, Il6st, Khsrp, Mars and Rara. Among these 12 genes, 8 (Add1, Camk2g, Cstb, Dr1, Epas1, Gna11, Hif1a, Khsrp) were already reduced in Ngb-Tg neurons compared to WT under rest conditions. Additionally, three genes that initially showed no changes in WT neurons (Ctgf, Egfr and Pea15) were downregulated after OGD in the Ngb-Tg neurons. These findings suggest that Ngb overexpression modulates mRNA expression of multiple hypoxic response genes in the early phase after OGD/reoxygenation. Further studies on these gene networks and interactions may lead to better understanding of Ngb in signaling pathways that contribute to neuroprotection.

Section snippets

Animals

All animal experiments were performed following protocols approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee in compliance with the NIH Guide for the Care and Use of Laboratory Animals.

Ngb-Tg mice and matched WT C57BL/6 mice were used to isolate cortical neuron cultures. We tried out best to minimize the number of animals used and their suffering.

Primary cortical neuronal culture

Primary neuronal culture was prepared from the cortex of embryonic day 15 mouse. In brief, the cortical

Ngb protein levels in WT and Ngb-Tg neurons, under normal condition or after oxygen–glucose deprivation followed by 4-h reoxygenation (O4/R4)

Total proteins were extracted from WT and Ngb-Tg neuron, either under normal condition, or after being treated by O4/R4. Western blotting was performed to examine Ngb protein levels as we previously described (Liu et al., 2009). Ngb protein level was slightly increased in WT neurons after O4/R4 (∼1.8-fold), but was significantly increased in Ngb-Tg neurons, either under normal condition (∼2.7-fold) or after O4/R4 (∼2.5-fold) (Fig. 1).

OGD-induced neurotoxicity was reduced in Ngb-Tg neurons compared to WT neurons

The neurotoxicity for WT or Ngb-Tg neurons was examined using

Discussion

Since the discovery of Ngb in 2000 (Burmester et al., 2000), experimental studies have documented that its overexpression is neuroprotective against hypoxia/ischemia (Garry and Mammen 2003, Khan et al 2006, Sun et al 2003, Wang et al 2008) and direct oxidative stress (Li et al 2008, Jin et al 2008). However, the neuroprotective mechanisms remain mysterious and not fully elucidated (Nienhaus and Nienhaus 2007, Brunori and Vallone 2006, Brunori and Vallone 2007, Greenberg et al 2008).

Ngb was

Conclusion

In summary, our results documented that Ngb overexpression alters the expression of a group of genes involved in hypoxia signaling pathways, suggesting this could at least partially contribute to mechanisms of Ngb's neuroprotective actions against hypoxia/OGD. Further studies to dissect these pathways will help us understand how Ngb works in brain, and this may ultimately reveal novel therapeutic approaches for stroke and other neurodegenerative disorders.

Acknowledgments

This work was supported in part by a NIH grant R01-NS049476 and a Scientist Development Grant 0435087 N from American Heart Association (to X.W.), and NIH grants R01-NS48422, R01-NS53560, and P01-NS55104 (to E.H.L.).

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Citation Excerpt :
Also in human neuroblastoma cell SH-SY5Y the Ngb overexpression protects against anoxia and glucose deprivation (Fordel et al., 2007b; Shao et al., 2009). Here, the time windows of cell death reported are in line with the idea that Ngb influences gene transcription, as cells die during the re-oxygenation phase, whereas in the early 4 hours no difference in cell death percentage between wild type and Ngb over-expressed cells has been detected (Yu et al., 2009b). In summary, Ngb seems to divert OGD-induced generalized gene downregulation while directing it to multiple cell survival mechanisms.
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Molecular NeuroscienceResearch PaperNeuroglobin-overexpression alters hypoxic response gene expression in primary neuron culture following oxygen glucose deprivation

Abstract

Section snippets

Animals

Primary cortical neuronal culture

Ngb protein levels in WT and Ngb-Tg neurons, under normal condition or after oxygen–glucose deprivation followed by 4-h reoxygenation (O4/R4)

OGD-induced neurotoxicity was reduced in Ngb-Tg neurons compared to WT neurons

Discussion

Conclusion

Acknowledgments

Biochem Biophys Res Commun

J Biol Chem

Lancet

J Biol Chem

Curr Opin Pharmacol

J Biol Chem

Neurosci Lett

Brain Res Mol Brain Res

Brain Res

Biochem Biophys Res Commun

Brain Res Mol Brain Res

Neuroscience

Biochem Biophys Res Commun

J Biol Chem

Biochem Biophys Res Commun

Neuron

Molecular Neuroscience
Research Paper
Neuroglobin-overexpression alters hypoxic response gene expression in primary neuron culture following oxygen glucose deprivation