Christian Ansgar Hundahl

Functional properties of neuroglobin and cytoglobin. Insights into the ancestral physiological roles of globins

Neuroglobin and cytoglobin are two recently discovered vertebrate globins, which are expressed at low levels in neuronal tissues and in all tissues investigated so far, respectively. Based on their amino acid sequences, these globins appear to be phylogenetically ancient and to have mutated less during evolution in comparison to the other vertebrate globins, myoglobin and hemoglobin. As with some plant and bacterial globins, neuroglobin and cytoglobin hemes are hexacoordinate in the absence of external ligands, in that the heme iron atom coordinates both a proximal and a distal His residue. While the physiological role of hexacoordinate globins is still largely unclear, neuroglobin appears to participate in the cellular defence against hypoxia. We present the current knowledge on the functional properties of neuroglobin and cytoglobin, and describe a mathematical model to evaluate the role of mammalian retinal neuroglobin in supplying O2 supply to the mitochondria. As shown, the model argues against a significant such role for neuroglobin, that more likely plays a role to scavenge reactive oxygen and nitrogen species that are generated following brain hypoxia. The O2 binding properties of cytoglobin, which is upregulated upon hypoxia, are consistent with a role for this protein in O2‐requiring reactions, such as those catalysed by hydroxylases. IUBMB Life, 56: 689‐696, 2004

Anatomical characterization of cytoglobin and neuroglobin mRNA and protein expression in the mouse brain.

The present study aimed at characterizing the anatomical and subcellular localization of cytoglobin (Cygb) and neuroglobin (Ngb) in the mouse brain by use of in situ hybridisation, immunohistochemistry and immunoelectron microscopy. Cygb and Ngb were only found in distinct brain areas and often in the same areas. We found intense staining in the piriform cortex, amygdala, hypothalamus (medial preoptic area, supra chiasmatic nucleus, lateral hypothalamus (LH), ventromedial hypothalamic nucleus, and the arcuate nucleus, habenular nuclei, laterodorsal tegmental nucleus (LDTg), pedunculopontine tegmental nucleus (PPTg), locus coeruleus, nucleus of the solitary tract and the spinal trigeminal nucleus. In addition Cygb is found in the hippocampus, the reticular thalamic nucleus, and the dorsal raphe nucleus; Ngb is found in the sub parabrachial nucleus. Co-localization of Cygb and Ngb is mainly observed in the LDTg and PPTg. Cygb and Ngb were found in cytoplasm, along neurotubuli, in mitochondria and in the nucleus by use of immunoelectron microscopy. Most neuronal nitric oxide synthase (nNOS)-positive neurons were found to co-localize Cygb, although not all nNOS neurones contain Cygb. Ngb co-localize with almost all orexin neurons in the LH. In conclusion the distribution of Cygb and Ngb seems much more restricted and coherent than previously reported. We believe other functions than pure oxygen buffers and neuroprotectants should be considered. The anatomical data indicate a role in NO signalling for Cygb and involvement in sleep-wake cycling for Cygb and Ngb.

Does neuroglobin protect neurons from ischemic insult? A quantitative investigation of neuroglobin expression following transient MCAo in spontaneously hypertensive rats.

Neuroglobin (NGB) is a recently characterized heme globin expressed primarily in retinal nerve cells and at very low levels in endocrine-active regions of vertebrate brains. When artificially over-expressed, NGB reduces the infarct size observed after transient Middle Cerebral Artery occlusion (tMCAo) in rats. This study addresses the post-ischemic NGB expression in vivo. Ten Spontaneously Hypertensive Rats (SHRs) were randomized to tMCAo (n = 6) or sham (n = 4), and euthanized 24 h later. NGB mRNA expression was determined by means of quantitative Reverse Transcription Polymerase Reaction (qRT-PCR). Thirteen animals subjected to either 90 min tMCAo (n = 7) or sham (n = 6) surgery, were euthanized 1 week after surgery. Post-ischemic expression of NGB and the neuronal marker NeuN was studied using free-floating immunohistochemistry. Design-based stereological quantification of NGB- and NeuN-positive cells in the striatum was performed using the optical fractionator. Significantly less NGB mRNA was expressed in the ischemic hemispheres of tMCAo animals after 24 h (P < or = 0.002). At the protein level, we found a significantly lower number of NGB- and NeuN-positive striatal neurons in tMCAo rats (P < or = 0.004). NGB expression was mainly confined to the hypothalamus and amygdala. Less than one out of every two thousand neurons expressed NGB in the striatum. In the ischemic territory we did not observe selective sparing of NGB expressing neurons. No significant change in the NGB/NeuN ratio was observed. Our data indicate that endogenous expressed NGB does not provide protection against ischemic injury induced by tMCAo in SHRs.

Effects of short-term hypoxia on neuroglobin levels and localization in mouse brain tissues

Nerve cells are highly susceptible to ischemic and hypoxic injuries. The neuroglobin (Ngb), found in vertebrate nerve cells, has been suggested to protect nerve cells from ischemic episodes by a yet unknown mechanism. However, contradicting reports exist regarding localization and up‐regulation of Ngb in response to hypoxia. The aim of the present study was to probe the distribution of Ngb proteins in mouse brain and retina by immunohistochemistry, and to quantify the levels of Ngb mRNA by reverse‐transcription‐polymerase chain reaction (RT‐PCR) after short‐term (2 h) exposure to 7.6% oxygen. We found Ngb to be present throughout the neocortex, most abundantly in the perirhinal, entorhinal and temporal cortical areas, the thalamus and hypothalamus, the choroid plexus, the olfactory bulb and the cranial nerve nuclei in the brainstem. Intense staining was observed in the mesencephalic central grey area and the Purkinje cells. Two‐hour hypoxic exposure caused no detectable changes in staining intensity or spatial distribution of Ngb neither in the Purkinje cells nor in any other brain areas observed. The RT‐PCR data supported the lack of differences in brain Ngb levels between normal and oxygen‐deprived animals. In the retina, Ngb localization by immunohistochemistry was confined to the inner segments of the photoreceptors, the plexiform layers and the ganglion cells. Short‐termed hypoxia did not change retinal Ngb levels as assessed by both techniques. The lack of Ngb up‐regulation in the brain is consistent with results from previous long‐term hypoxic experiments, suggesting that Ngb is not regulated by pure hypoxia in vivo.

A biophysical signature of network affiliation and sensory processing in mitral cells

One defining characteristic of the mammalian brain is its neuronal diversity. For a given region, substructure, layer or even cell type, variability in neuronal morphology and connectivity persists. Although it is well known that such cellular properties vary considerably according to neuronal type, the substantial biophysical diversity of neurons of the same morphological class is typically averaged out and ignored. Here we show that the amplitude of hyperpolarization-evoked sag of membrane potential recorded in olfactory bulb mitral cells is an emergent, homotypic property of local networks and sensory information processing. Simultaneous whole-cell recordings from pairs of cells show that the amount of hyperpolarization-evoked sag potential and current (Ih) is stereotypic for mitral cells belonging to the same glomerular circuit. This is corroborated by a mosaic, glomerulus-based pattern of expression of the HCN2 (hyperpolarization-activated cyclic nucleotide-gated channel 2) subunit of the Ih channel. Furthermore, inter-glomerular differences in both membrane potential sag and HCN2 protein are diminished when sensory input to glomeruli is genetically and globally altered so that only one type of odorant receptor is universally expressed. Population diversity in this intrinsic property therefore reflects differential expression between local mitral cell networks processing distinct odour-related information.

Neuroglobin-Deficiency Exacerbates Hif1A and c-FOS Response, but Does Not Affect Neuronal Survival during Severe Hypoxia In Vivo

Background Neuroglobin (Ngb), a neuron-specific globin that binds oxygen in vitro, has been proposed to play a key role in neuronal survival following hypoxic and ischemic insults in the brain. Here we address whether Ngb is required for neuronal survival following acute and prolonged hypoxia in mice genetically Ngb-deficient (Ngb-null). Further, to evaluate whether the lack of Ngb has an effect on hypoxia-dependent gene regulation, we performed a transcriptome-wide analysis of differential gene expression using Affymetrix Mouse Gene 1.0 ST arrays. Differential expression was estimated by a novel data analysis approach, which applies non-parametric statistical inference directly to probe level measurements. Principal Findings Ngb-null mice were born in expected ratios and were normal in overt appearance, home-cage behavior, reproduction and longevity. Ngb deficiency had no effect on the number of neurons, which stained positive for surrogate markers of endogenous Ngb-expressing neurons in the wild-type (wt) and Ngb-null mice after 48 hours hypoxia. However, an exacerbated hypoxia-dependent increase in the expression of c-FOS protein, an immediate early transcription factor reflecting neuronal activation, and increased expression of Hif1A mRNA were observed in Ngb-null mice. Large-scale gene expression analysis identified differential expression of the glycolytic pathway genes after acute hypoxia in Ngb-null mice, but not in the wts. Extensive hypoxia-dependent regulation of chromatin remodeling, mRNA processing and energy metabolism pathways was apparent in both genotypes. Significance According to these results, it appears unlikely that the loss of Ngb affects neuronal viability during hypoxia in vivo. Instead, Ngb-deficiency appears to enhance the hypoxia-dependent response of Hif1A and c-FOS protein while also altering the transcriptional regulation of the glycolytic pathway. Bioinformatic analysis of differential gene expression yielded novel predictions suggesting that chromatin remodeling and mRNA metabolism are among the key regulatory mechanisms when adapting to prolonged hypoxia.

Neuroglobin in the Rat Brain (II): Co-Localisation with Neurotransmitters

In an accompanying article, we found that neuroglobin (Ngb) was expressed in a few well-defined nuclei in the rat brain. Here, we show by use of immunohistochemistry and in situ hybridisation (ISH) that Ngb co-localise with several specific neurotransmitters. Ngb co-localise consistently with tyrosine hydroxylase (TH) in the noradrenergic/adrenergic A1/C1 and A2/C2; the noradrenergic A5, A6 and A7. Ngb were not observed to co-localise TH in the dopaminergic A8-A16 cell populations. Ngb were only seen to co-localise with choline acetyltransferase (ChAT) in the laterodorsal tegmental nucleus (LDTg) and in the pontine tegmental nucleus (PPTg). Many Ngb-ir neurones co-localised with neuronal nitric oxide synthase (nNOS) in the LDTg, whereas fewer Ngb-ir neurones co-localise nNOS in the anterior basomedial (BMA) and the posterodorsal medial (MePD) amygdaloid nucleus, in the medial preoptic area (MPA) and in part of the lateral hypothalamus (LH). Ngb-ir neurones co-localise heme oxygenase 1 (HO-1) in the LDTg and locus coeruleus. Ngb-ir neurones co-localise hypocretin-1 (Hcrt1) in the perifornical (PeF) and perifornical lateral hypothalamus (PeFLH). Within the LH, Ngb-ir neurones co-localised melanin concentration hormone (MCH). A few Ngb-ir perikarya in the paraventricular hypothalamic nucleus (PVN) co-localised arginine vasopressin (aVP). Ngb were not observed to co-localise with serotonin, vasointestinal peptide (VIP), or cocaine amphetamine-regulated transcript (CART) at any places. In the present study, we found no evidence that one or more particular neurotransmitters are coupled 100% to Ngb or that Ngb is coupled 100% to a specific neurotransmitter. Based on these findings, we suggest that Ngb could be involved in some sort of regulation of the sleep-wake cycle. Secondly, that Ngb in some neurones is involved in regulation of gaseous neurotransmission, and that this in any given case only involves a subset of neurones. To us this indicates that the cellular and physiological function of Ngb in different subsets of neurones might not be identical, or that all neurones containing Ngb has one thing in common that we at presently not are aware of.

Neuroglobin in the Rat Brain : Localization

Neuroglobin (Ngb) is a neuronal hemeprotein similar to myoglobin and hemoglobin and shares their capability for oxygen binding. It has thus been proposed that Ngb acts as an oxygen reservoir or combats reactive oxygen species. In the present study, we investigated the Ngb expression pattern in the rat brain using immunohistochemistry, in situ hybridization, and quantitative real-time PCR (qRT-PCR). This revealed the interesting finding that Ngb expression is restricted to a few neurone populations, many of which are involved in the sleep-wake cycle, circadian regulation or food regulation. In the forebrain we found intense Ngb expression in neurones in the piriform cortex, the central and medial amygdala, the medial preoptic area, the suprachiasmatic nucleus (SCN), the hypothalamic paraventricular nucleus, the perifornical nucleus, the lateral hypothalamus. Within the mid- and hindbrain Ngb expressing neurones were found in the laterodorsal tegmental nucleus, the pedunculo pontine tegmental nucleus, the locus coeruleus, and the lateral parabrachial nucleus. In the medulla oblongata Ngb expressing neurones were found in the nucleus of the solitary tract. The qRT-PCR data showed a diurnal variation of Ngb mRNA in the SCN, having a peak in the day time (light-period) and nadir during night (dark-period).

Circadian Behaviour in Neuroglobin Deficient Mice

Neuroglobin (Ngb), a neuron-specific oxygen-binding globin with an unknown function, has been proposed to play a key role in neuronal survival. We have previously shown Ngb to be highly expressed in the rat suprachiasmatic nucleus (SCN). The present study addresses the effect of Ngb deficiency on circadian behavior. Ngb-deficient and wild-type (wt) mice were placed in running wheels and their activity rhythms, endogenous period and response to light stimuli were investigated. The effect of Ngb deficiency on the expression of Period1 (Per1) and the immediate early gene Fos was determined after light stimulation at night and the neurochemical phenotype of Ngb expressing neurons in wt mice was characterized. Loss of Ngb function had no effect on overall circadian entrainment, but resulted in a significantly larger phase delay of circadian rhythm upon light stimulation at early night. A light-induced increase in Per1, but not Fos, gene expression was observed in Ngb-deficient mice. Ngb expressing neurons which co-stored Gastrin Releasing Peptide (GRP) and were innervated from the eye and the geniculo-hypothalamic tract expressed FOS after light stimulation. No PER1 expression was observed in Ngb-positive neurons. The present study demonstrates for the first time that the genetic elimination of Ngb does not affect core clock function but evokes an increased behavioural response to light concomitant with increased Per1 gene expression in the SCN at early night.

The Nerve Hemoglobin of the Bivalve Mollusc Spisula solidissima MOLECULAR CLONING, LIGAND BINDING STUDIES, AND PHYLOGENETIC ANALYSIS

Members of the hemoglobin (Hb) superfamily are present in nerve tissue of several vertebrate and invertebrate species. In vertebrates they display hexacoordinate heme iron atoms and are typically expressed at low levels (μm). Their function is still a matter of debate. In invertebrates they have a hexa- or pentacoordinate heme iron, are mostly expressed at high levels (mm), and have been suggested to have a myoglobin-like function. The native Hb of the surf clam, Spisula solidissima, composed of 162 amino acids, does not show specific deviations from the globin templates. UV-visible and resonance Raman spectroscopy demonstrate a hexacoordinate heme iron. Based on the sequence analogy, the histidine E7 is proposed as a sixth ligand. Kinetic and equilibrium measurements show a moderate oxygen affinity (P50 ∼0.6 torr) and no cooperativity. The histidine binding affinity is 100-fold lower than in neuroglobin. Phylogenetic analysis demonstrates a clustering of the S. solidissima nerve Hb with mollusc Hbs and myoglobins, but not with the vertebrate neuroglobins. We conclude that invertebrate nerve Hbs expressed at high levels are, despite the hexacoordinate nature of their heme iron, not essentially different from other intracellular Hbs. They most likely fulfill a myoglobin-like function and enhance oxygen supply to the neurons.