Neuroglobin protects the brain from experimental stroke in vivo

Abstract

Neuroglobin (Ngb) is an O2-binding protein localized to cerebral neurons of vertebrates, including humans. Its physiological role is unknown but, like hemoglobin, myoglobin, and cytoglobin͞histo-globin, it may transport O 2, detoxify reactive oxygen species, or serve as a hypoxia sensor. We reported recently that hypoxia stimulates transcriptional activation of Ngb in cultured cortical neurons and that antisense inhibition of Ngb expression increases hypoxic neuronal injury, whereas overexpression of Ngb confers resistance to hypoxia. These findings are consistent with a role for Ngb in promoting neuronal survival after hypoxic insults in vitro. Here we report that in rats, intracerebroventricular administration of an Ngb antisense, but not sense, oligodeoxynucleotide increases infarct volume and worsens functional neurological outcome, whereas intracerebral administration of a Ngb-expressing adenoassociated virus vector reduces infarct size and improves functional outcome, after focal cerebral ischemia induced by occlusion of the middle cerebral artery. We conclude that Ngb acts as an endogenous neuroprotective factor in focal cerebral ischemia and may therefore represent a target for the development of new treatments for stroke.T he ability to sense and respond to hypoxia is a universal attribute of eukaryotic cells, but the role of this ability in protecting cells from ischemic insults and its potential for therapeutic application are unclear. One prominent feature of cellular adaptation to hypoxia or ischemia consists of the increased expression of hypoxia-inducible proteins (1), including proteins with the capacity to protect the brain from ischemic insults (2, 3). Examples include hypoxia-inducible factor-1 (4), erythropoietin (5), vascular endothelial growth factor (6), heme oxygenase-1 (7), and adrenomedullin (8). These proteins are likely to exert their neuroprotective effects through diverse mechanisms but their hypoxia-responsiveness depends ultimately on O 2 -binding proteins that can sense hypoxia and trigger appropriate cellular adaptations (9, 10).The globins are a family of heme proteins that can bind, transport, scavenge, detoxify, and sense gases like O 2 and NO (11). Four vertebrate globins have been identified. Hemoglobin, which differs from other vertebrate globins in occurring as a tetramer, is localized to erythrocytes and transports O 2 between the lungs and other tissues. Myoglobin (Mgb) is monomeric and is localized to the cytoplasm of skeletal and cardiac myocytes. Cytoglobin or histoglobin (12, 13) is expressed widely in both neural and nonneural vertebrate tissues, but its functions are unknown. The recent discovery of neuroglobin (Ngb; ref. 14), which is expressed primarily in cerebral neurons (15), is induced by neuronal hypoxia and cerebral ischemia (16) and protects neurons from hypoxia in vitro (16), suggests that this protein may have a role in sensing or responding to neuronal hypoxia, which could have implications for the pathophysiology and treatment of stroke.To test the hy...

A neuroglobin-overexpressing transgenic mouse

“…The observation that Ngb, an O 2 -binding heme protein expressed in cerebral neurons (Burmester et al, 2000), is induced by neuronal hypoxia and cerebral ischemia and protects neurons from hypoxia and ischemia (Sun et al, 2001;Sun et al, 2003), suggests that this protein may have a role in sensing or responding to neuronal hypoxia. Because O 2 is critical for cellular functions like mitochondrial oxidative phosphorylation, cells require mechanisms for sensing hypoxia, transducing this signal, and eliciting adaptive cellular responses (Bunn and Poyton, 1996).…”

“…The observation that Ngb expression is induced by hypoxia, cobalt and deferoxamine (Sun et al, 2001) is consistent with, but not proof of, induction via HIF-1, which would suggest a more downstream, hypoxia-responding, role for Ngb. The ability of Ngb overexpression to protect neurons from hypoxia (Sun et al, 2001) and ischemia (Sun et al, 2003;Khan et al, 2006) could be explained by enhancement of either hypoxia sensing or hypoxia responding. The transgenic model described here may be useful for resolving these and other controversies about Ngb.…”

“…These brain regions were chosen for screening because of our interest in possible effects of Ngb overexpression on focal cerebral ischemia in the middle cerebral artery distribution (Sun et al, 2003). However, Ngb overexpression was also observed in other brain areas, including olfactory bulb, hippocampus, cerebellum and brainstem.…”

“…Fluorescence immunohistochemistry was performed as described in detail elsewhere (Sun et al, 2001;Sun et al, 2003). Primary antibodies were goat polyclonal anti-Ngb (1:200; Santa Cruz Biotechnology), mouse monoclonal anti-NeuN (1:200; Chemicon) and mouse monoclonal anti-GFAP (1:150; Sigma), and secondary antibodies were fluorescein isothiocyanate (FITC)-conjugated rat-absorbed donkey anti-mouse and rhodamine-conjugated donkey anti-goat immunoglobulin G (Jackson ImmunoResearch;1:200).…”

“…In vivo studies yielded similar results. Thus, the volume of cerebral infarcts caused by occlusion of the middle cerebral artery in the rat was increased by intracerebroventricular administration of a Ngb antisense oligodeoxynucleotide and reduced by intracerebral injection of a Ngbexpressing adeno-associated virus vector (Sun et al, 2003).…”