Critical re-evaluation of neuroglobin expression reveals conserved patterns among mammals.

Neuroglobin (Ngb) is a respiratory protein that is almost exclusively expressed in the vertebrate nervous system. Despite many years of research, the exact function and even the expression sites of Ngb are still a matter of debate. However, to investigate hypotheses surrounding the potential roles of Ngb, a detailed knowledge of its major and minor expression sites is indispensable. We have therefore evaluated Ngb expression by extensive bioinformatic analysis using publicly available transcriptome data (RNA-Seq). During mammalian brain development, we observed low embryonic expression of Ngb mRNA and an increase after birth, arguing against a role of Ngb in fetal hypoxia tolerance. In adult mouse brain, we found highest Ngb mRNA levels in the hypothalamus, where expression was up to 100-fold stronger than in cerebral cortex, cerebellum or hippocampus, as confirmed by qRT-PCR and Western blotting. High Ngb expression in the hypothalamus was found conserved in humans and other mammals. Thus, Ngb mRNA is expressed at a basal level in many mammalian brain regions, but shows distinctive regional peaks. RNA-Seq analysis further revealed only low levels of Ngb mRNA in retina and testes and no signal in standard tumor cell lines, thus raising questions concerning previous studies and functional hypotheses. In conclusion, this broad-scale expression study may point to distinct Ngb functions for high- and low-expressing cells and tissues and argues against a single, generic role of Ngb as an oxygen supplier or as an endogenous protectant in all nerve cells.

Neuroglobin and cytoglobin overexpression protects human SH-SY5Y neuroblastoma cells against oxidative stress-induced cell death.

Although reactive oxygen species (ROS) at physiological concentrations are required for normal cell function, excessive production of ROS is detrimental to cells. Neuroglobin and cytoglobin are two globins, whose functions are still a matter of debate. A potential role in the detoxification of ROS is suggested. The influence of neuroglobin and cytoglobin on cell death after oxidative stress in human neuroblastoma SH-SY5Y cells was evaluated. Exposure of SH-SY5Y cells to paraquat or H(2)O(2) resulted in a concentration- and time-dependent induction of apoptotic and necrotic cell death. H(2)O(2) was 16 times more potent to induce cell death as compared to paraquat. SH-SY5Y cells transfected with plasmid DNA containing the neuroglobin or cytoglobin sequence showed enhanced survival after exposure to 300 microM H(2)O(2) for 24h as compared to untransfected controls. This finding suggests that neuroglobin and cytoglobin protect SH-SY5Y cells against oxidative stress-induced cell death.

Divergent distribution of cytoglobin and neuroglobin in the murine eye.

Neuroglobin (Ngb) and cytoglobin (Cygb) are two vertebrate globins with yet poorly defined functions. Previous studies had demonstrated a high expression level of neuroglobin in the mammalian retina, being in line with a respiratory function. Here we show that in the mouse eye, cytoglobin is localised in fibroblasts of the ciliary processes and the choroidea. In the neuronal retina, cytoglobin is expressed in a subset of neurons of the ganglion cell and inner nuclear layers. Cytoglobin is also present in the inner plexiform layer, but absent from the pigment cells. Neuroglobin is localised in photoreceptor inner segments, the plexiform layers and the ganglion cell layer. The divergent distribution of neuroglobin and cytoglobin in the mammalian retina suggests distinct functions of these proteins in the vertebrate nervous system. While neuroglobin seems to be associated with oxygen consumption, a respiratory function of cytoglobin is unlikely.

Neuroglobin and cytoglobin in search of their role in the vertebrate globin family.

Neuroglobin and cytoglobin are two recent additions to the family of heme-containing respiratory proteins of man and other vertebrates. Here, we review the present state of knowledge of the structures, ligand binding kinetics, evolution and expression patterns of these two proteins. These data provide a first glimpse into the possible physiological roles of these globins in the animal's metabolism. Both, neuroglobin and cytoglobin are structurally similar to myoglobin, although they contain distinct cavities that may be instrumental in ligand binding. Kinetic and structural studies show that neuroglobin and cytoglobin belong to the class of hexa-coordinated globins with a biphasic ligand-binding kinetics. Nevertheless, their oxygen affinities resemble that of myoglobin. While neuroglobin is evolutionarily related to the invertebrate nerve-globins, cytoglobin shares a more recent common ancestry with myoglobin. Neuroglobin expression is confined mainly to brain and a few other tissues, with the highest expression observed in the retina. Present evidence points to an important role of neuroglobin in neuronal oxygen homeostasis and hypoxia protection, though other functions are still conceivable. Cytoglobin is predominantly expressed in fibroblasts and related cell types, but also in distinct nerve cell populations. Much less is known about its function, although in fibroblasts it might be involved in collagen synthesis.

Neuron-specific expression of neuroglobin in mammals.

Neuroglobin, a vertebrate oxygen-binding protein, is expressed in many regions of the adult brain. We examined the cell type-specific expression of neuroglobin in neurons and astroglial cells in primary cultures of fetal hippocampal cells and sections of the adult mouse brain using neuroglobin-specific polyclonal antibodies and cell type-specific markers NeuN and GFAP to differentiate between neurons and glial cells. Neuroglobin is exclusively expressed in neurons, but not in astroglial cells. Accordingly, neuroglobin was detected in two neuroblastoma cell lines (N2a, SH-SY5Y) and the pheochromocytoma cell line PC-12, but not in glioblastoma cell lines (DKMG, GAMG) or other, non-neural cells (HeLa, Vero). Analysis of the neuroglobin genomic sequence from man and mouse identifies sequence motifs with similarity to the neuron-restrictive silencer element, possibly explaining a neuron-specific expression of neuroglobin.

Cytoglobin is a respiratory protein in connective tissue and neurons, which is up-regulated by hypoxia.

Cytoglobin is a recently discovered vertebrate globin distantly related to myoglobin, and its function is unknown. Here we present the first detailed analysis of the distribution and expression of cytoglobin. Northern and Western blotting experiments show the presence of cytoglobin mRNA and protein in a broad range of tissues. Quantitative PCR demonstrates an up-regulation of cytoglobin mRNA levels in rat heart and liver under hypoxic conditions (22 and 44 h of 9% oxygen). Immunofluorescence studies with three antibodies directed against different epitopes of the protein consistently show cytoglobin in connective tissue fibroblasts as well as in hepatic stellate cells. Cytoglobin is also present in chondroblasts and osteoblasts and shows a decreased level of expression upon differentiation to chondrocytes and osteocytes. Cytoglobin is located in the cytoplasm of these cell types. Evidence against an exclusively nuclear localization of cytoglobin, as recently proposed, is also provided by transfection assays with green fluorescent protein fusion constructs, which demonstrates the absence of an active nuclear import. The differential expression of cytoglobin argues against a general respiratory function of this molecule, but rather indicates a connective tissue-specific function. We hypothesize that cytoglobin may be involved in collagen synthesis. Cytoglobin expression was also observed in some neuronal subpopulations of the central and the peripheral nervous systems. Surprisingly, cytoglobin is localized in both the cytoplasm and nucleus of neurons, indicating a possible additional role of this protein in neuronal tissues.

The cellular and subcellular localization of neuroglobin and cytoglobin -- a clue to their function?

Neuroglobin and cytoglobin are recently discovered respiratory proteins of vertebrates with yet ill-defined physiological functions. Neuroglobin is widely expressed in neurons, but not glia, in the vertebrate central and peripheral nervous systems. Other major expression sites are the retina and endocrine tissues. This distribution is indicative of a function of neuroglobin in metabolically most active, oxygen-consuming cell types, but does not yet allow to safely distinguish between different cellular roles, such as oxygen homeostasis, scavenging of reactive oxygen species or sustaining energy metabolism. Cytoglobin is predominantly expressed in connective tissue fibroblasts and related cell types in the body organs. Its main function may therefore be related to the specific amounts of extracellular matrix. Cytoglobin may hypothetically be involved in the oxygen-consuming maturation of collagen proteins. Cytoglobin is also expressed in distinct cell types of brain and retina. Its distribution strikingly differs from neuroglobin, suggesting an independent, yet unknown function.

Localization of neuroglobin protein in the mouse brain.

Neuroglobin is a recently discovered vertebrate oxygen-binding respiratory protein. In situ hybridization data demonstrated that neuroglobin-mRNA is widely expressed in neuronal cells of the central and peripheral nervous systems as well as in endocrine cells. The present study was conducted to investigate the presence of neuroglobin protein in neurons of the mouse brain. A polyclonal antibody directed against a synthetic peptide of neuroglobin was raised in rabbits and affinity-purified. The specificity of the antibody was demonstrated by ELISA and preabsorption tests. We report here for the first time that neuroglobin is expressed on the protein level in many brain sites including cerebral cortical regions, subcortical structures such as thalamus and hypothalamus, nuclei of cranial nerves in the brainstem and cerebellum. Thus, the widespread distribution of neuroglobin protein is in good agreement with its mRNA localization. Regionally differing intensities of immunostaining suggest different levels of neuroglobin protein expression, in line with the idea that brain regions show variation in their tolerance towards hypoxic conditions.

How does the eye breathe? Evidence for neuroglobin-mediated oxygen supply in the mammalian retina.

Visual performance of the vertebrate eye requires large amounts of oxygen, and thus the retina is one of the highest oxygen-consuming tissues of the body. Here we show that neuroglobin, a neuron-specific respiratory protein distantly related to hemoglobin and myoglobin, is present at high amounts in the mouse retina (approximately 100 microm). The estimated concentration of neuroglobin in the retina is thus about 100-fold higher than in the brain and is in the same range as that of myoglobin in the muscle. Neuroglobin is expressed in all neurons of the retina but not in the retinal pigment epithelium. Neuroglobin mRNA was detected in the perikarya of the nuclear and ganglion layers of the neuronal retina, whereas the protein was present mainly in the plexiform layers and in the ellipsoid region of photoreceptor inner segment. The distribution of neuroglobin correlates with the subcellular localization of mitochondria and with the relative oxygen demands, as the plexiform layers and the inner segment consume most of the retinal oxygen. These findings suggest that neuroglobin supplies oxygen to the retina, similar to myoglobin in the myocardium and the skeletal muscle.