Senescent Secretome of Blind Mole Rat Spalax Inhibits Malignant Behavior of Human Breast Cancer Cells Triggering Bystander Senescence and Targeting Inflammatory Response.

Subterranean blind mole rat, Spalax, has developed strategies to withstand cancer by maintaining genome stability and suppressing the inflammatory response. Spalax cells undergo senescence without the acquisition of senescence-associated secretory phenotype (SASP) in its canonical form, namely, it lacks the main inflammatory mediators. Since senescence can propagate through paracrine factors, we hypothesize that conditioned medium (CM) from senescent Spalax fibroblasts can transmit the senescent phenotype to cancer cells without inducing an inflammatory response, thereby suppressing malignant behavior. To address this issue, we investigated the effect of CMs of Spalax senescent fibroblasts on the proliferation, migration, and secretory profile in MDA-MB-231 and MCF-7 human breast cancer cells. The results suggest that Spalax CM induced senescence in cancer cells, as evidenced by increased senescence-associated beta-galactosidase (SA-ß-Gal) activity, growth suppression and overexpression of senescence-related p53/p21 genes. Contemporaneously, Spalax CM suppressed the secretion of the main inflammatory factors in cancer cells and decreased their migration. In contrast, human CM, while causing a slight increase in SA-ß-Gal activity in MDA-MB-231 cells, did not decrease proliferation, inflammatory response, and cancer cell migration. Dysregulation of IL-1α under the influence of Spalax CM, especially the decrease in the level of membrane-bound IL1-α, plays an important role in suppressing inflammatory secretion in cancer cells, which in turn leads to inhibition of cancer cell migration. Overcoming of SASP in tumor cells in response to paracrine factors of senescent microenvironment or anti-cancer drugs represents a promising senotherapeutic strategy in cancer treatment.

Cloning of two splice variants of Spalax heparanase encoding for truncated proteins.

Heparanase is an endoglycosidase that degrades heparan sulfate side chains of heparan sulfate-proteoglycans. It liberates heparan sulfate-bound growth factors and thereby promotes blood vessel sprouting and angiogenesis. The subterranean blind mole rat, Spalax, is a wild mammal that lives most of its life in underground tunnels where it experiences sharp fluctuations in oxygen and carbon dioxide levels. We described two splice variants of heparanase from Spalax, Splice 7 and splice 36, both devoid of heparanase enzymatic activity. Splice 7 increases tumor growth, while splice 36 functions as a dominant negative to wild-type heparanase and decreases tumor growth and metastasis. Here, we describe two novel splice variants of Spalax heparanase, splice 67 and splice 612. These splice variants result in production of a shorter heparanase proteins that are similar to the wild-type native heparanase in their N-terminal but have unique C-terminals. Both splice 67 and 612 lack heparan sulfate degradation activity.

The transcriptome landscape of the carcinogenic treatment response in the blind mole rat: insights into cancer resistance mechanisms.

BACKGROUND: Spalax, the blind mole rat, developed an extraordinary cancer resistance during 40 million years of evolution in a subterranean, hypoxic, thus DNA damaging, habitat. In 50 years of Spalax research, no spontaneous cancer development has been observed. The mechanisms underlying this resistance are still not clarified. We investigated the genetic difference between Spalax and mice that might enable the Spalax relative resistance to cancer development. We compared Spalax and mice responses to a treatment with the carcinogen 3-Methylcholantrene, as a model to assess Spalax' cancer-resistance. RESULTS: We compared RNA-Seq data of untreated Spalax to Spalax with a tumor and identified a high number of differentially expressed genes. We filtered these genes by their expression in tolerant Spalax that resisted the 3MCA, and in mice, and found 25 genes with a consistent expression pattern in the samples susceptible to cancer among species. Contrasting the expressed genes in Spalax with benign granulomas to those in Spalax with malignant fibrosarcomas elucidated significant differences in several pathways, mainly related to the extracellular matrix and the immune system. We found a central cluster of ECM genes that differ greatly between conditions. Further analysis of these genes revealed potential microRNA targets. We also found higher levels of gene expression of some DNA repair pathways in Spalax than in other murines, like the majority of Fanconi Anemia pathway. CONCLUSION: The comparison of the treated with the untreated tissue revealed a regulatory complex that might give an answer how Spalax is able to restrict the tumor growth. By remodeling the extracellular matrix, the possible growth is limited, and the proliferation of cancer cells was potentially prevented. We hypothesize that this regulatory cluster plays a major role in the cancer resistance of Spalax. Furthermore, we identified 25 additional candidate genes that showed a distinct expression pattern in untreated or tolerant Spalax compared to animals that developed a developed either a benign or malignant tumor. While further study is necessary, we believe that these genes may serve as candidate markers in cancer detection.

Adipose-Derived Stem Cells of Blind Mole Rat Spalax Exhibit Reduced Homing Ability: Molecular Mechanisms and Potential Role in Cancer Suppression.

Adipose-derived stem cells (ADSCs) are recruited by cancer cells from the adjacent tissue, and they become an integral part of the tumor microenvironment. Here, we report that ADSCs from the long-living, tumor-resistant blind mole rat, Spalax, have a low ability to migrate toward cancer cells compared with cells from its Rattus counterpart. Tracking 5-ethynyl-2'-deoxyuridine (EdU)-labeled ADSCs, introduced to tumor-bearing nude mice, toward the xenografts, we found that rat ADSCs intensively migrated and penetrated the tumors, whereas only a few Spalax ADSCs reached the tumors. Moreover, rat ADSCs, but not Spalax ADSCs, acquired endothelial-like phenotype and incorporated in the intratumoral reticular structure resembling a vasculature. Likewise, endothelial-like cells differentiated from Spalax and rat ADSCs could form capillary-like structures; however, the tube densities were higher in rat-derived cells. Using time-lapse microscopy, in vitro wound-healing, and transwell migration assays, we demonstrated the impaired motility and low polarization ability of Spalax ADSCs. To assess whether the phosphorylated status of myosin light chain (MLC) is involved in the decreased motility of Spalax ADSCs, we inhibited MLC phosphorylation by blocking of Rho-kinase (ROCK). Inhibition of ROCK resulted in the suppression of MLC phosphorylation, acquisition of actin polarization, and activation of motility and migration of Spalax ADSCs. We propose that reduced ADSCs migration to cancer and poor intratumoral angiogenesis play a role in Spalax's cancer resistance. Learning more about the molecular strategy of noncancerous cells in Spalax to resist oncogenic stimuli and maintain a nonpermissive tumor milieu may lead us to developing new cancer-preventive strategy in humans. Stem Cells 2018;36:1630-1642.

Postnatal Exocrine Pancreas Growth by Cellular Hypertrophy Correlates with a Shorter Lifespan in Mammals.

Developmental processes in different mammals are thought to share fundamental cellular mechanisms. We report a dramatic increase in cell size during postnatal pancreas development in rodents, accounting for much of the increase in organ size after birth. Hypertrophy of pancreatic acinar cells involves both higher ploidy and increased biosynthesis per genome copy; is maximal adjacent to islets, suggesting endocrine to exocrine communication; and is partly driven by weaning-related processes. In contrast to the situation in rodents, pancreas cell size in humans remains stable postnatally, indicating organ growth by pure hyperplasia. Pancreatic acinar cell volume varies 9-fold among 24 mammalian species analyzed, and shows a striking inverse correlation with organismal lifespan. We hypothesize that cellular hypertrophy is a strategy for rapid postnatal tissue growth, entailing life-long detrimental effects.

Hypoxia tolerance, longevity and cancer-resistance in the mole rat Spalax - a liver transcriptomics approach.

The blind subterranean mole rat Spalax shows a remarkable tolerance to hypoxia, cancer-resistance and longevity. Unravelling the genomic basis of these adaptations will be important for biomedical applications. RNA-Seq gene expression data were obtained from normoxic and hypoxic Spalax and rat liver tissue. Hypoxic Spalax broadly downregulates genes from major liver function pathways. This energy-saving response is likely a crucial adaptation to low oxygen levels. In contrast, the hypoxia-sensitive rat shows massive upregulation of energy metabolism genes. Candidate genes with plausible connections to the mole rat's phenotype, such as important key genes related to hypoxia-tolerance, DNA damage repair, tumourigenesis and ageing, are substantially higher expressed in Spalax than in rat. Comparative liver transcriptomics highlights the importance of molecular adaptations at the gene regulatory level in Spalax and pinpoints a variety of starting points for subsequent functional studies.

Genome maintenance and bioenergetics of the long-lived hypoxia-tolerant and cancer-resistant blind mole rat, Spalax: a cross-species analysis of brain transcriptome.

The subterranean blind mole rat, Spalax, experiences acute hypoxia-reoxygenation cycles in its natural subterranean habitat. At the cellular level, these conditions are known to promote genomic instability, which underlies both cancer and aging. However, Spalax is a long-lived animal and is resistant to both spontaneous and induced cancers. To study this apparent paradox we utilized a computational procedure that allows detecting differences in transcript abundance between Spalax and the closely related above-ground Rattus norvegicus in individuals of different ages. Functional enrichment analysis showed that Spalax whole brain tissues maintain significantly higher normoxic mRNA levels of genes associated with DNA damage repair and DNA metabolism, yet keep significantly lower mRNA levels of genes involved in bioenergetics. Many of the genes that showed higher transcript abundance in Spalax are involved in DNA repair and metabolic pathways that, in other species, were shown to be downregulated under hypoxia, yet are required for overcoming replication- and oxidative-stress during the subsequent reoxygenation. We suggest that these differentially expressed genes may prevent the accumulation of DNA damage in mitotic and post-mitotic cells and defective resumption of replication in mitotic cells, thus maintaining genome integrity as an adaptation to acute hypoxia-reoxygenation cycles.

Adaptive patterns in the p53 protein sequence of the hypoxia- and cancer-tolerant blind mole rat Spalax.

BACKGROUND: The subterranean blind mole rat, Spalax (genus Nannospalax) endures extreme hypoxic conditions and fluctuations in oxygen levels that threaten DNA integrity. Nevertheless, Spalax is long-lived, does not develop spontaneous cancer, and exhibits an outstanding resistance to carcinogenesis in vivo, as well as anti-cancer capabilities in vitro. We hypothesized that adaptations to similar extreme environmental conditions involve common mechanisms for overcoming stress-induced DNA damage. Therefore, we aimed to identify shared features among species that are adapted to hypoxic stress in the sequence of the tumor-suppressor protein p53, a master regulator of the DNA-damage response (DDR). RESULTS: We found that the sequences of p53 transactivation subdomain 2 (TAD2) and tetramerization and regulatory domains (TD and RD) are more similar among hypoxia-tolerant species than expected from phylogeny. Specific positions in these domains composed patterns that are more frequent in hypoxia-tolerant species and have proven to be good predictors of species' classification into stress-related categories. Some of these positions, which are known to be involved in the interactions between p53 and critical DDR proteins, were identified as positively selected. By 3D modeling of p53 interactions with the coactivator p300 and the DNA repair protein RPA70, we demonstrated that, compared to humans, these substitutions potentially reduce the binding of these proteins to Spalax p53. CONCLUSIONS: We conclude that extreme hypoxic conditions may have led to convergent evolutionary adaptations of the DDR via TAD2 and TD/RD domains of p53.

Non-image Forming Light Detection by Melanopsin, Rhodopsin, and Long-Middlewave (L/W) Cone Opsin in the Subterranean Blind Mole Rat, Spalax Ehrenbergi: Immunohistochemical Characterization, Distribution, and Connectivity.

The blind mole rat, Spalax ehrenbergi, can, despite severely degenerated eyes covered by fur, entrain to the daily light/dark cycle and adapt to seasonal changes due to an intact circadian timing system. The present study demonstrates that the Spalax retina contains a photoreceptor layer, an outer nuclear layer (ONL), an outer plexiform layer (OPL), an inner nuclear layer (INL), an inner plexiform layer (IPL), and a ganglion cell layer (GCL). By immunohistochemistry, the number of melanopsin (mRGCs) and non-melanopsin bearing retinal ganglion cells was analyzed in detail. Using the ganglion cell marker RNA-binding protein with multiple splicing (RBPMS) it was shown that the Spalax eye contains 890 ± 62 RGCs. Of these, 87% (752 ± 40) contain melanopsin (cell density 788 melanopsin RGCs/mm(2)). The remaining RGCs were shown to co-store Brn3a and calretinin. The melanopsin cells were located mainly in the GCL with projections forming two dendritic plexuses located in the inner part of the IPL and in the OPL. Few melanopsin dendrites were also found in the ONL. The Spalax retina is rich in rhodopsin and long/middle wave (L/M) cone opsin bearing photoreceptor cells. By using Ctbp2 as a marker for ribbon synapses, both rods and L/M cone ribbons containing pedicles in the OPL were found in close apposition with melanopsin dendrites in the outer plexus suggesting direct synaptic contact. A subset of cone bipolar cells and all photoreceptor cells contain recoverin while a subset of bipolar and amacrine cells contain calretinin. The calretinin expressing amacrine cells seemed to form synaptic contacts with rhodopsin containing photoreceptor cells in the OPL and contacts with melanopsin cell bodies and dendrites in the IPL. The study demonstrates the complex retinal circuitry used by the Spalax to detect light, and provides evidence for both melanopsin and non-melanopsin projecting pathways to the brain.

Molecular evolution of antioxidant and hypoxia response in long-lived, cancer-resistant blind mole rats: The Nrf2-Keap1 pathway.

The Nrf2-Keap1 pathway is crucial for the cellular antioxidant and hypoxia response in vertebrates. Deciphering its modifications in hypoxia-adapted animals will help understand its functionality under environmental stress and possibly allow for knowledge transfer into biomedical research. The blind mole rat Spalax, a long-lived cancer-resistant rodent, lives in burrows underground and is adapted to severely hypoxic conditions. Here we have conducted a bioinformatical survey of Spalax core genes from the Nrf2-Keap1 pathway on the coding sequence level in comparison to other hypoxia-tolerant and -sensitive rodents. We find strong sequence conservation across all genes, illustrating the pathway's importance. One of the central players however, Spalax Keap1, shows a non-conservative amino acid substitution from tyrosine to cysteine in its intervening region (IVR) domain. Cysteines in this location have been shown to be of high functional relevance to the binding and degradation of Nrf2. Therefore, this peculiar substitution could influence the cellular Nrf2 levels in Spalax and, thereby, downstream gene expression in the antioxidant pathway, contributing to the special adaptive phenotype of the blind mole rat.

Genome-wide adaptive complexes to underground stresses in blind mole rats Spalax.

The blind mole rat (BMR), Spalax galili, is an excellent model for studying mammalian adaptation to life underground and medical applications. The BMR spends its entire life underground, protecting itself from predators and climatic fluctuations while challenging it with multiple stressors such as darkness, hypoxia, hypercapnia, energetics and high pathonecity. Here we sequence and analyse the BMR genome and transcriptome, highlighting the possible genomic adaptive responses to the underground stressors. Our results show high rates of RNA/DNA editing, reduced chromosome rearrangements, an over-representation of short interspersed elements (SINEs) probably linked to hypoxia tolerance, degeneration of vision and progression of photoperiodic perception, tolerance to hypercapnia and hypoxia and resistance to cancer. The remarkable traits of the BMR, together with its genomic and transcriptomic information, enhance our understanding of adaptation to extreme environments and will enable the utilization of BMR models for biomedical research in the fight against cancer, stroke and cardiovascular diseases.

They live in the land down under: thyroid function and basal metabolic rate in the Blind Mole Rat, Spalax.

The Israeli blind subterranean mole rat (Spalax ehrenbergi superspecies) lives in sealed underground burrows under extreme, hypoxic conditions. The four Israeli Spalax allospecies have adapted to different climates, the cool-humid (Spalax galili, 2 n = 52 chromosomes), semihumid (S. golani, 2 n = 54) north regions, warm-humid (S. carmeli, 2 n = 58) central region and the warm-dry S. judaei, 2 n = 60) southern regions. A dramatic interspecies decline in basal metabolic rate (BMR) from north to south, even after years of captivity, indicates a genetic basis for this BMR trait. We examined the possibility that the genetically-conditioned interspecies BMR difference was expressed via circulating thyroid hormone. An unexpected north to south increase in serum free thyroxine (FT4) and total 3, 5, 3'-triiodo-L-thyronine (T3) (p < 0.02) correlated negatively with previously published BMR measurements. The increases in serum FT4 and T3 were symmetrical, so that the T3:FT4 ratio - interpretable as an index of conversion of T4 to T3 in nonthyroidal tissues - did not support relative decrease in production of T3 as a contributor to BMR. Increased north-to-south serum FT4 and T3 levels also correlated negatively with hemoglobin/hematocrit. North-to-south adaptations in spalacids include decreased BMR and hematocrit/hemoglobin in the face of increasing thyroid hormone levels, arguing for independent control of hormone secretion and BMR/hematocrit/hemoglobin. But the significant inverse relationship between thyroid hormone levels and BMR/hematocrit/hemoglobin is also consistent with a degree of cellular resistance to thyroid hormone action that protects against hormone-induced increase in oxygen consumption in a hostile, hypoxic environment.

Pronounced cancer resistance in a subterranean rodent, the blind mole-rat, Spalax: in vivo and in vitro evidence.

BACKGROUND: Subterranean blind mole rats (Spalax) are hypoxia tolerant (down to 3% O2), long lived (>20 years) rodents showing no clear signs of aging or aging related disorders. In 50 years of Spalax research, spontaneous tumors have never been recorded among thousands of individuals. Here we addressed the questions of (1) whether Spalax is resistant to chemically-induced tumorigenesis, and (2) whether normal fibroblasts isolated from Spalax possess tumor-suppressive activity. RESULTS: Treating animals with 3-Methylcholantrene (3MCA) and 7,12-Dimethylbenz(a) anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA), two potent carcinogens, confirmed Spalax high resistance to chemically induced cancers. While all mice and rats developed the expected tumors following treatment with both carcinogens, among Spalax no tumors were observed after DMBA/TPA treatment, while 3MCA induced benign fibroblastic proliferation in 2 Spalax individuals out of12, and only a single animal from the advanced age group developed malignancy 18 months post-treatment. The remaining animals are still healthy 30 months post-treatment. In vitro experiments showed an extraordinary ability of normal Spalax cultured fibroblasts to restrict malignant behavior in a broad spectrum of human-derived and in newly isolated Spalax 3MCA-induced cancer cell lines. Growth of cancer cells was inhibited by either direct interaction with Spalax fibroblasts or with soluble factors released into culture media and soft agar. This was accompanied by decreased cancer cell viability, reduced colony formation in soft agar, disturbed cell cycle progression, chromatin condensation and mitochondrial fragmentation. Cells from another cancer resistant subterranean mammal, the naked mole rat, were also tested for direct effect on cancer cells and, similar to Spalax, demonstrated anti-cancer activity. No effect on cancer cells was observed using fibroblasts from mouse, rat or Acomys. Spalax fibroblast conditioned media had no effect on proliferation of noncancerous cells. CONCLUSIONS: This report provides pioneering evidence that Spalax is not only resistant to spontaneous cancer but also to experimentally induced cancer, and shows the unique ability of Spalax normal fibroblasts to inhibit growth and kill cancer cells, but not normal cells, either through direct fibroblast-cancer cell interaction or via soluble factors. Obviously, along with adaptation to hypoxia, Spalax has evolved efficient anti-cancer mechanisms yet to be elucidated. Exploring the molecular mechanisms allowing Spalax to survive in extreme environments and to escape cancer as well as to kill homologous and heterologous cancer cells may hold the key for understanding the molecular nature of host resistance to cancer and identify new anti-cancer strategies for treating humans.

Transcription pattern of p53-targeted DNA repair genes in the hypoxia-tolerant subterranean mole rat Spalax.

The tumor suppressor gene p53 induces growth arrest and/or apoptosis in response to DNA damage/hypoxia. Inactivation of p53 confers a selective advantage to tumor cells under a hypoxic microenvironment during tumor progression. The subterranean blind mole rat, Spalax, spends its life underground at low-oxygen tensions, hence developing a wide range of respiratory/molecular adaptations to hypoxic stress, including critical changes in p53 structure and signaling pathway. The highly conserved p53 Arg(R)-172 is substituted by lysine (K) in Spalax, identical with a tumor-associated mutation. Functionality assays revealed that Spalax p53 is unable to activate apoptotic target genes but is still capable of activating cell cycle arrest genes. Furthermore, we have shown that the transcription patterns of representative p53-induced genes (Apaf1 and Mdm2) in Spalax are influenced by hypoxia. Cell cycle arrest allows the cells to repair DNA damage via different DNA repair genes. We tested the transcription pattern of three p53-related DNA repair genes (p53R2, Mlh1, and Msh2) under normoxia and short-acute hypoxia in Spalax, C57BL/6 wild-type mice, and two strains of mutant C57BL/6 mice, each carrying a different mutation at the R172 position. Our results show that while wild-type/mutant mice exhibit strong hypoxia-induced reductions of repair gene transcript levels, no such inhibition is found in Spalax under hypoxia. Moreover, unlike mouse p53R2, Spalax p53R2 transcript levels are strongly elevated under hypoxia. These results suggest that critical repair functions, which are known to be inhibited under hypoxia in mice, remain active in Spalax, as part of its unique hypoxia tolerance mechanisms.

Transcriptome analysis of the spalax hypoxia survival response includes suppression of apoptosis and tight control of angiogenesis.

BACKGROUND: The development of complex responses to hypoxia has played a key role in the evolution of mammals, as inadequate response to this condition is frequently associated with cardiovascular diseases, developmental disorders, and cancers. Though numerous studies have used mice and rats in order to explore mechanisms that contribute to hypoxia tolerance, these studies are limited due to the high sensitivity of most rodents to severe hypoxia. The blind subterranean mole rat Spalax is a hypoxia tolerant rodent, which exhibits unique longevity and therefore has invaluable potential in hypoxia and cancer research. RESULTS: Using microarrays, transcript abundance was measured in brain and muscle tissues from Spalax and rat individuals exposed to acute and chronic hypoxia for varying durations. We found that Spalax global gene expression response to hypoxia differs from that of rat and is characterized by the activation of functional groups of genes that have not been strongly associated with the response to hypoxia in hypoxia sensitive mammals. Using functional enrichment analysis of Spalax hypoxia induced genes we found highly significant overrepresentation of groups of genes involved in anti apoptosis, cancer, embryonic/sexual development, epidermal growth factor receptor binding, coordinated suppression and activation of distinct groups of transcription factors and membrane receptors, in addition to angiogenic related processes. We also detected hypoxia induced increases of different critical Spalax hub gene transcripts, including antiangiogenic genes associated with cancer tolerance in Down syndrome human individuals. CONCLUSIONS: This is the most comprehensive study of Spalax large scale gene expression response to hypoxia to date, and the first to use custom Spalax microarrays. Our work presents novel patterns that may underlie mechanisms with critical importance to the evolution of hypoxia tolerance, with special relevance to medical research.

Cross talk between S-nitrosylation and S-glutathionylation in control of the Na,K-ATPase regulation in hypoxic heart.

Oxygen-induced regulation of Na,K-ATPase was studied in rat myocardium. In rat heart, Na,K-ATPase responded to hypoxia with a dose-dependent inhibition in hydrolytic activity. Inhibition of Na,K-ATPase in hypoxic rat heart was associated with decrease in nitric oxide (NO) production and progressive oxidative stress. Accumulation of oxidized glutathione (GSSG) and decrease in NO availability in hypoxic rat heart were followed by a decrease in S-nitrosylation and upregulation of S-glutathionylation of the catalytic α-subunit of the Na,K-ATPase. Induction of S-glutathionylation of the α-subunit by treatment of tissue homogenate with GSSG resulted in complete inhibition of the enzyme in rat a myocardial tissue homogenate. Inhibitory effect of GSSG in rat sarcolemma could be significantly decreased upon activation of NO synthases. We have further tested whether oxidative stress and suppression of the Na,K-ATPase activity are observed in hypoxic heart of two subterranean hypoxia-tolerant blind mole species (Spalax galili and Spalax judaei). In both hypoxia-tolerant Spalax species activity of the enzyme and tissue redox state were maintained under hypoxic conditions. However, localization of cysteines within the catalytic subunit of the Na,K-ATPase was preserved and induction of S-glutathionylation by GSSG in tissue homogenate inhibited the Spalax ATPase as efficiently as in rat heart. The obtained data indicate that oxygen-induced regulation of the Na,K-ATPase in the heart is mediated by a switch between S-glutathionylation and S-nitrosylation of the regulatory thiol groups localized at the catalytic subunit of the enzyme.

Hypoxia associated NMDA receptor 2 subunit composition: developmental comparison between the hypoxia-tolerant subterranean mole-rat, Spalax, and the hypoxia-sensitive rat.

Vertebrate brains are sensitive to oxygen depletion, which may lead to cell death. Hypoxia sensitivity originates from the high intrinsic rate of ATP consumption of brain tissue, accompanied by the release of glutamate, leading to the opening of ionotropic glutamate receptors, such as N-methyl-D-aspartate (NMDA) receptors (NMDARs). The relative expression levels of the four NMDAR-2 (NR2) subunits change during mammalian development with higher levels of units NR2B and NR2D observed during early development and correlated with hypoxic tolerance during embryonic and neonatal stages of development. Higher levels of NR2D are also abundant in brains of hypoxia tolerant species such as the crucian carp. The subterranean mole-rat, Spalax spends its life underground in sealed burrows and has developed a wide range of adaptations to this special niche including hypoxia-tolerance. In this study, we compared the in vivo mRNA expression of NR2 subunits in the brains of embryonic, neonatal and adult Spalax and rat. Our results demonstrate that under normoxic conditions, mRNA levels of NR2D are higher in Spalax than in rat at all developmental stages studied and are similar to levels in neonatal rat and in other hypoxia/anoxia tolerant species. Furthermore, under hypoxia Spalax NR2D mRNA levels increase while no response was observed in rat. Similarly, hypoxia induces an increase in mRNA levels of Spalax NR2A, claimed to promote neuronal survival. We suggest that indeed the proportional combinations of NMDAR-2 subunits contribute to the ability of the Spalax brain to cope with hypoxic environments.

Living with stress: regulation of antioxidant defense genes in the subterranean, hypoxia-tolerant mole rat, Spalax.

Lack of oxygen is life threatening for most mammals. It is therefore of biomedical interest to investigate the adaptive mechanisms which enable mammalian species to tolerate extremely hypoxic conditions. The subterranean mole rat Spalax survives substantially longer periods of hypoxia than the laboratory rat. We hypothesized that genes of the antioxidant defense, detoxifying harmful reactive oxygen species generated during hypoxia and hyperoxia, are involved in Spalax underground adaptation. Using quantitative RT-PCR, we analyzed the mRNA expression levels of seven antioxidant defense genes (catalase, glutathione peroxidase 1, glutathione-S-transferase Pi1, heme oxygenase 1, superoxide dismutase 1 and 2) and a master regulator of this stress pathway, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in several tissues of two Israeli Spalax species, S. galili (2n=52) and S. judaei (2n=60), and rat. We also studied the differential expression of these genes after experimental hypoxia and hyperoxia as oxidative stress treatments. We found that mRNA levels and transcriptional responses are species and tissue specific. There are constitutively higher transcript levels of antioxidant genes and their transcription factor Nrf2 in Spalax tissue as compared to rat, suggesting an increased ability in the mole rat to withstand hypoxic/hyperoxic insults. In contrast to Spalax, the rat reacts to experimental oxidative stress by changes in gene regulation. In addition, Spalax Nrf2 reveals unique amino acid changes, which may be functionally important for this transcription factor and indicate positive (Darwinian) selection. Antioxidant defense genes are therefore important targets for adaptive change during evolution of hypoxia tolerance in Spalax.

Methionine sulfoxide reductases and methionine sulfoxide in the subterranean mole rat (Spalax): characterization of expression under various oxygen conditions.

The blind subterranean mole rat (Spalax ehrenbergi) exhibits a relatively long life span, which is attributed to an efficient antioxidant defense affording protection against accumulation of oxidative modifications of proteins. Methionine residues can be oxidized to methionine sulfoxide (MetO) and then enzymatically reduced by the methionine sulfoxide reductase (Msr) system. In the current study we have isolated the cDNA sequences of the Spalax Msr genes as well as 23 additional selenoproteins and monitored the activities of Msr enzymes in liver and brain of rat (Rattus norvegicus), Spalax galili, and Spalax judaei under normoxia, hypoxia, and hyperoxia. Under normoxia, the Msr activity was lower in S. galili in comparison to S. judaei and R. norvegicus especially in the brain. The pattern of Msr activity of the three species was similar throughout the tested conditions. However, exposure of the animals to hypoxia caused a significant enhancement of Msr activity, especially in S. galili. Hyperoxic exposure showed a highly significant induction of Msr activity compared with normoxic conditions for R. norvegicus and S. galili brain. It was concluded that among all species examined, S. galili appears to be more responsive to oxygen tension changes and that the Msr system is upregulated mainly by severe hypoxia.