A substantial number of studies have investigated Ngb expression and its potential neuroprotective properties during hypoxia and ischemia (for review please see 
). Studies in rodents have reported lack of Ngb regulation 
, down-regulation 
and up-regulation of Ngb 
following hypoxia. Ischemic rodent models have also yielded conflicting results ranging from no differential expression and no neuroprotective properties in relation to ischemia 
to up-regulation 
and significant neuroprotective properties when Ngb expression was modulated by viral gene transfer 
or up-regulated by transgenic means 
. In spite of numerous studies on the relation of Ngb and hypoxia/ischemia there remains a lack of consensus as to the role of Ngb protein in neuroprotection. Therefore, in order to increase our understanding of Ngb's potential therapeutic role, we created genetically Ngb-deficient mice and studied their response to hypoxia. We hypothesized that Ngb-deficient mice would be more susceptible to the adverse effects of hypoxia if Ngb protein had substantial neuroprotective properties. The study specifically evaluated the effect of Ngb deficiency on neuronal survival and on transcriptional regulation following acute and prolonged hypoxia.
Ngb-null mice are born in expected rations, they have normal survival to adulthood (age 8 weeks) and they show no obvious differences in overt appearance, body weight and behavior when compared to wt littermates. Hence, Ngb does not seem to be critical for normal development and vitality. To evaluate the effect of Ngb deficiency on neuronal viability after hypoxia, we used Orexin-A-positive and Cygb-positive neurons as surrogate markers for Ngb neurons in the lateral hypothalamus and hindbrain, respectively. We found no statistical difference in the number of these neurons between wt and Ngb-null mice in normoxia or after 24 and 48 hours of hypoxia, suggesting that the loss of Ngb protein does not affect neuronal viability even after severe prolonged hypoxia. However, the apparent lack of cell death does not imply that Ngb-positive and Ngb-deficient neurons are equally resistant to hypoxia. Therefore, we investigated the expression of cleaved caspase-3, a marker of activated apoptotic effector mechanisms, in wt and Ngb-null hypoxic mice. No cleaved caspase-3 immunoreactivity was detected in any of the mice indicating that Ngb deficiency is not sufficient to activate this apoptotic effector molecule during hypoxia. These results are contradictory to reports of decreased survival of Ngb-deficient cells after hypoxia in vitro
and to the reports on the increase in ischemic infarct size after adenovirus-mediated down-regulation of Ngb in vivo 
. It is important to note however, that the in vivo
hypoxic conditions differ markedly from both the hypoxia models in the cell culture and from the models of cerebral ischemia. Specifically, the oxygen concentration in the microenvironment surrounding neurons during in vivo
experimental hypoxia is unknown while intact cerebral blood flow still provides cells with nutrients and serum, which is not the case in ischemia. In addition, one cannot exclude that inborn deficiency in Ngb function might induce compensatory mechanisms, which are able to substitute for the lack of Ngb during normal and hypoxic conditions. In the aforementioned studies the experimental down-regulation of Ngb was acute, which is unlikely to allow for a substituting effect. However, since the mice used in the present study had no prior experience of hypoxia there seems to be little reason to assume that the brains of Ngb-null mice had adapted to low oxygen availability. It is therefore more likely that Ngb is not necessary for neuronal survival even after severe prolonged hypoxia.
Although Ngb deficiency does not seem to affect neuronal survival during hypoxia, we found a widespread and more pronounced increase in the number of c-FOS-IR neurons in Ngb-null mice than in wt mice after acute hypoxia. A hypothesis that Ngb deficiency lowers the threshold for hypoxia-induced gene expression response is also supported by the global analysis of gene expression, which identified the increased expression of Hif1A and its heterodimer partner Arnt in Ngb-null mice after 24 hours of hypoxia.
Our results from the large-scale analysis of gene expression are well in line with previous reports (see 
, for a review) and they confirmed the hypoxia-dependent regulation of pathways related to, for example, apoptosis, cell growth (“mTOR signaling”), synthesis of ATP (“oxidative phosphorylation”) and angiogenesis (“VEGF signaling pathway”) in both genotypes. Based on a broader categorization of the differentially regulated pathways we will focus the discussion on glucose metabolism, chromatin remodeling and RNA processing.
Most cells produce ATP by mitochondrial oxidative phosphorylation under normoxic conditions. However, oxidative phosphorylation is inefficient under hypoxic conditions, and adaptation to hypoxic stress is achieved by the down-regulation of mitochondrial oxygen consumption and by increased reliance on anaerobic glycolysis for energy production 
. Consequently, the largest functional group of genes regulated by HIF1 in many cell types are associated with glucose metabolism 
. We identified a coordinated down-regulation of genes encoding subunits of mitochondrial ATP synthase complexes F0 and F1, several cytochrome c oxidase subunits, NADH dehydrogenase subunits and an up-regulation of glucose transporter 1 (Slc2a1) gene in both genotypes after 24-h hypoxia (Table S15
). Interestingly, an up-regulation of monocarboxylate transporter 4 gene (Slc16a4) encoding a plasma membrane protein that removes lactate, the end product of anaerobic glycolysis, from the cell, was detected after exposure to hypoxia only in Ngb-null mice. More surprisingly, a down-regulation of the following genes of the glycolysis pathway was detected in Ngb-null mice after acute hypoxia: Eno1 (enolase 1, alpha non-neuron), Eno2 (enolase 2, gamma neuronal), Gapdh (glyceraldehyde-3-phosphate dehydrogenase), Pfkl (phosphofructokinase, liver, B-type Gene) and Aldoa (aldolase A, fructose-bisphosphate). Of those, Gapdh was down-regulated also after 24 hours hypoxia. Recent evidence indicating that hypoxia-dependent post-translational protein modification by SUMO1 (small ubiquitin related modifier 1) may activate the glycolytic pathway 
, prompted us to look at the differential expression of the 3 genes of the SUMO protein family and SUMO/sentrin specific protease genes, which encode proteins which can reverse SUMOylation 
. We detected an up-regulation of Sumo3 and Sumo2 after acute hypoxia in the wt and Ngb-deficient mice, respectively. Sumo1 and Sumo2 were up-regulated in the wt mice, but not in the Ngb-deficient mice after 24-h hypoxia. Down-regulation of Senp8 (SUMO/sentrin specific protease family member 8) was detected in wt mice after both acute and 24-h hypoxia. These observations point to a more widespread hypoxia-dependent regulation of the SUMOylation pathway genes in the wt mice than in the Ngb-deficient mice. Altogether, the observations cited above suggest that the transcriptional down-regulation of mitochondrial activity during 24 hours hypoxia is intact in Ngb-null mice, but the regulation of the glycolytic pathway and SUMO-related genes is altered.
Hypoxia-dependent regulation of chromatin remodeling pathways was evident from the down-regulation of genes with tags “nucleosome assembly” and “Phosphorylation of histone H2AX at Serine-139 by ATM” in both genotypes after acute hypoxia and in Ngb-null mice also after 24 hours hypoxia. In contrast, there was an up-regulation of genes with tags “chromatin modification” and “Association of HMGB1/HMGB2 with chromatin” after 24 hours hypoxia in both genotypes. Inspection of the implicated genes (Table S16
) revealed numerous core histones from histone cluster 1 and 2 genes, ATP-dependent chromatin remodelers (Smarca4, Smarca5, Chd4, Chd7, Chd8), genes of heterochromatin- (Cbx3) and euchromatin-associated proteins (Hmgb1, Hmgb2, Hmgb3, Hmgn3, Pole3), histone methyl transferases (Mll5, Setdb2, Ehmt1), histone demethylases (Jmjd6, Kdm2a, Kdm3a, Kdm4a, Kdm4c, Kdm5a, Kdm6a) and RNA polymerase 2 associated factors (Paf1, Leo1) etc. Although studies on hypoxia-dependent chromatin remodeling are rather scarce 
, hypoxia-dependent modification of histones has been documented in a number of cell lines 
. Our results demonstrate the down-regulation of core histone genes after acute hypoxia and the up-regulation of genes encoding histone-modifying proteins after 24 hours hypoxia in both genotypes. Incidentally, a study of endothelial nitric oxide gene has documented the eviction of histones from its core promoter and a subsequent reincorporation of histones with altered post-translational modifications in response to hypoxia 
. It is possible that a similar hypoxia-dependent resetting of histone modification patterns occurs in the brain and that it revolves in stages, which coincide with the down-regulation of core histones, followed by the up-regulation of histone modification pathways. The importance of histones in adapting to a hypoxic environment is also suggested by a comparative exome-wide study of SNP frequencies in ethnic Tibetans where 3 out of the 30 most significant loci were linked to genes in histone cluster 1 
. Finally, among the 60 transcripts with most reliable response to hypoxia, we found several which encode proteins regulating chromatin structure (Hist3h2a, H2afj, Chd7 and Hmgb2).
Up-regulation of mRNA processing and mRNA metabolic pathways (tags “Formation and Maturation of mRNA Transcript”, “mRNA metabolic process”, “mRNA processing”, “mRNA Splicing”) was detected in both genotypes after 24 hours hypoxia, but not after acute hypoxia. The main post-transcriptional mechanisms which regulate gene expression during hypoxia appear to be mRNA turnover and translational control 
. We detected, for example, the up-regulation of Ptbp2 (known to promote Hif1a mRNA translation and stabilize Vegf mRNA during hypoxia, see 
for overview), genes of several heterogeneous nuclear ribonucleoproteins (regulate translation by affecting mRNA secondary structure), various RNA binding motif proteins (related mostly to mRNA splicing) and THO complex genes (required for normal transcriptional elongation and recruitment of splicing factors) 
. These observations suggest that post-transcriptional processes might play a prominent role in the adaptation of the brain tissue to prolonged hypoxia.
The present study demonstrates that the lack of neuroglobin in mice alters the response of c-FOS, Hif1A and the regulation of the glycolytic pathway genes whereas there is no effect on neuronal and organismal survival rate or behavior during severe acute and prolonged hypoxia. Analysis of global gene expression in both genotypes suggests chromatin remodeling and mRNA metabolism to be among the key regulatory mechanisms when adapting to prolonged hypoxia. Further studies are necessary to clarify the role of neuroglobin in the regulation of cellular metabolism.