Neuroglobin, cytoglobin, and myoglobin contribute to hypoxia adaptation of the subterranean mole rat Spalax Aaron Avivia,1,2, Frank Gerlachb,1, Alma Joela, Stefan Reussc, Thorsten Burmesterd, Eviatar Nevoa,2, and Thomas Hankelnb,2 aInstitute of Evolution, University of Haifa, Mount Carmel, Haifa 31905, Israel; bInstitute of Molecular Genetics, Johannes Gutenberg-University Mainz, D-55099 Mainz, Germany; cInstitute of Microanatomy and Neurobiology, School of Medicine, Johannes Gutenberg-University Mainz, D-55131 Mainz, Germany; and dZoological Institute and Museum, Biocenter Grindel, University of Hamburg, D-20146 Hamburg, Germany Contributed by Eviatar Nevo, November 1, 2010 (sent for review July 2, 2010) The subterranean mole rat Spalax is an excellent model for studying with the hypoxia-sensitive rodent Rattus norvegicus, Spalax survives adaptation of a mammal toward chronic environmental hypoxia. substantially longer at low ambient O2 levels and high CO2 without Neuroglobin (Ngb) and cytoglobin (Cygb) are O2-binding respiratory serious deleterious effects or behavioral changes (6). proteins and thus candidates for being involved in molecular hyp- Hypoxia tolerance mechanisms identified in Spalax as com- oxia adaptations of Spalax. Ngb is expressed primarily in vertebrate pared with R. norvegicus include blood properties, anatomical nerves, whereas Cygb is found in extracellular matrix-producing and biochemical changes in respiratory organs (2, 4), and dif- cells and in some neurons. The physiological functions of both pro- ferences in the structure and function of a growing list of gene products (7–10). Transcription patterns of genes related to hyp- teins are not fully understood but discussed with regard to O2 sup- fi ply, the detoxification of reactive oxygen or nitrogen species, and oxic stress differ interspeci cally in Spalax (5, 11) and between Spalax and rat, involving key players such as erythropoietin (Epo) apoptosis protection. Spalax Ngb and Cygb coding sequences are and its receptors, and hypoxia-inducible factor-1α (Hif-1α) (12, strongly conserved. However, mRNA and protein levels of Ngb in 13). An important adaptation of Spalax to hypoxic habitats is Spalax brain are 3-fold higher than in Rattus norvegicus under nor- mediated by an increased blood vessel density, which is triggered EVOLUTION moxia. Importantly, Spalax expresses Ngb in neurons and addition- by a constitutively higher expression (compared with rat) of vas- ally in glia, whereas in hypoxia-sensitive rodents Ngb expression is cular endothelial growth factor (Vegf), Hif-1α, and HuR, a post- limited to neurons. Hypoxia causes an approximately 2-fold down- transcriptional stabilizer of Vegf mRNA (6, 14). regulation of Ngb mRNA in brain of rat and mole rat. A parallel The aerobic metabolism of mammals relies on respiratory regulatory response was found for myoglobin (Mb) in Spalax and proteins that function in the delivery and storage of O2.Hbin rat muscle, suggesting similar functions of Mb and Ngb. Cygb also erythrocytes transports O2 from the lungs to inner organs (15). revealed an augmented normoxic expression in Spalax vs. rat brain, Myoglobin (Mb) in cardiac and striated muscles acts as a local O2 fi but not in heart or liver, indicating distinct tissue-speci c functions. store and facilitates intracellular diffusion of O2 (16). Ten years Hypoxia induced Cygb transcription in heart and liver of both mam- ago, neuroglobin (Ngb) and cytoglobin (Cygb) were discovered mals, with the most prominent mRNA up-regulation (12-fold) in Spalax as unique members of the mammalian globin family (17). The heart. Our data suggest that tissue globins contribute to the re- physiological functions of Ngb and Cygb are still uncertain. In markable tolerance of Spalax toward environmental hypoxia. This most mammals, Ngb resides in neurons of the central and pe- is consistent with the proposed cytoprotective effect of Ngb and ripheral nervous systems, as well as in endocrine organs (18, 19). Cygb under pathological hypoxic/ischemic conditions in mammals. Ngb may have an Mb-like role in supplying O2 to the mitochon- drial respiratory chain (18, 20, 21). Alternatively, it may function as a scavenger of reactive oxygen or nitrogen species (ROS/RNS) xygen levels that are inadequate to sustain cellular energy (22, 23) or protect cells from cytochrome c-induced apoptosis (24, Oproduction constitute a life-threatening condition for mam- 25). Regardless of its ultimate role, there is conclusive evidence mals. Metabolically most active tissues (e.g., nerve cells) are ex- that Ngb localization is tightly linked to active oxidative metab- quisitely sensitive to a reduction of O2 (hypoxia), and humans are olism and mitochondria (19, 20). The highest Ngb level was found severely affected by hypoxic disease conditions like stroke or myo- in the neuronal retina, which also has the highest O -consuming fi 2 cardial ischemia. It is therefore mandatory to investigate the speci c rate in the body (20, 26). Several studies have shown that Ngb is adaptations evolved by mammals that live in naturally hypoxic cyto- and neuroprotective (27–29). Recently it was demonstrated environments where low ambient O2 tensions limit the availability that in the hooded seal, a diving mammal that tolerates prolonged of O2 to the organism (1). hypoxia of the brain (30), Ngb is expressed in astrocytes (31). This The blind mole rat Spalax spends its entire life in underground may indicate an unusual shift of oxidative metabolism from neu- burrows that can be extremely hypoxic/hypercapnic (2, 3). The rons to glial cells. Spalacidae, originating 25–40 million years ago, have evolved Cygb occurs predominantly in the fibroblast cell lineage, as physiological strategies enabling their respiratory and cardiovas- well as in some neurons (32–34). The function of Cygb is even cular systems to cope with hypoxia more efficiently than other less clear than that of Ngb but has been interpreted in terms of mammalian species (2, 4). The four karyotypically distinct allo- ROS defense or O2 supply to certain enzymes (17, 33, 35). species of Spalax in Israel are adapted to different climatic regimes. The strongest differences in ecological conditions are observed between Spalax galili (karyotype 2n = 52), inhabiting the northern Author contributions: A.A., F.G., and T.H. designed research; A.A., F.G., A.J., S.R., and T.B. cool-humid Upper Galilee Mountains with heavy soil, which often performed research; F.G. analyzed data; and A.A., S.R., T.B., E.N., and T.H. wrote the becomes flooded, and Spalax judaei (2n = 60), which reside in the paper. warm-dry south with light-aerated soil. The most efficient hypoxic The authors declare no conflict of interest. adaptation has consequently been demonstrated in S. galili,with 1A.A. and F.G. contributed equally to this work. higher normoxic breathing and heart rate as well as higher he- 2To whom correspondence may be addressed. E-mail: [email protected], matocrit and Hb levels as compared with S. judaei (2, 5). Another [email protected], or [email protected]. two Spalax species, Spalax golani (2n = 54) and Spalax carmeli This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (2n = 58), are intermediate in their hypoxia adaptation. Compared 1073/pnas.1015379107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1015379107 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 Globins are candidates that may enable Spalax to better survive low ambient oxygen conditions. To understand the particular role of Ngb, Cygb, and Mb in hypoxia tolerance, we have studied their sequences, expression patterns, and gene regulation in different Spalax species in comparison with rat. The data provide indirect evidence to the physiological function(s) of Ngb and Cygb in mam- mals and point to the biomedical significance of these proteins. Results Sequence Analysis of Spalax Ngb, Cygb, and Mb. Ngb and Cygb gene sequences from S. carmeli were reported previously (36). Here we have cloned, sequenced, and compared the coding regions of Ngb and Cygb from all four Spalax species (Fig. S1). As in other mam- mals (18, 37), the Spalax Ngb cDNA encodes a strongly conserved protein of 151 amino acids. The four Spalax species maximally differ by two amino acids, and the best match to other genera was found with mouse Ngb (94% identity, 96% similarity). Spalax Cygb cDNAs encode a protein of 190 amino acids, as typical for most other mammals. Cygb proteins of S. carmeli and S. galili are iden- tical and show two amino acid differences to S. golani and S. judaei, which has one additional substitution. S. golani Cygb is 95% iden- tical/99% similar to mouse Cygb. Only very few Spalax-specific amino acid replacements were observed for Ngb and Cygb (Fig. S1). Homology modeling showed that these substitutions are on the surface of the Ngb and Cygb proteins (Fig. S2). Except for the minor differences, Spalax Ngb and Cygb include the conserved sequence hallmarks of functional O2 carriers [e.g., the proximal and distal His (F8 and E7) and PheCD1, which are involved in heme and ligand binding]. The coding region of Spalax Mb (462 bp) translates into a protein of 154 amino acids. Its sequence—here obtained from S. carmeli (2n = 58)—matched the published S. judaei Mb pro- Fig. 1. Ngb immunofluorescence in Spalax brain. (A) Frontal section of tein sequence (38) with 98.1% identity. Again, all functional Spalax brain at the level of the lateral ventricle (LV) and anterior commissure positions are conserved, and the observed replacements are (ac). Ngb staining of neurons was found in the piriform (Pir), insular, sensory, found in other mammals as well. and motor regions of the cerebral cortex (CC) and in the striatum (caudate nucleus-putamen, CPu), as well as medial and basal forebrain regions. fi Expression Patterns of Ngb and Cygb in Spalax Tissue.