Alma Joel

Neuroglobin, cytoglobin, and myoglobin contribute to hypoxia adaptation of the subterranean mole rat Spalax

The subterranean mole rat Spalax is an excellent model for studying adaptation of a mammal toward chronic environmental hypoxia. Neuroglobin (Ngb) and cytoglobin (Cygb) are O2-binding respiratory proteins and thus candidates for being involved in molecular hypoxia adaptations of Spalax. Ngb is expressed primarily in vertebrate nerves, whereas Cygb is found in extracellular matrix-producing cells and in some neurons. The physiological functions of both proteins are not fully understood but discussed with regard to O2 supply, the detoxification of reactive oxygen or nitrogen species, and apoptosis protection. Spalax Ngb and Cygb coding sequences are strongly conserved. However, mRNA and protein levels of Ngb in Spalax brain are 3-fold higher than in Rattus norvegicus under normoxia. Importantly, Spalax expresses Ngb in neurons and additionally in glia, whereas in hypoxia-sensitive rodents Ngb expression is limited to neurons. Hypoxia causes an approximately 2-fold down-regulation of Ngb mRNA in brain of rat and mole rat. A parallel regulatory response was found for myoglobin (Mb) in Spalax and rat muscle, suggesting similar functions of Mb and Ngb. Cygb also revealed an augmented normoxic expression in Spalax vs. rat brain, but not in heart or liver, indicating distinct tissue-specific functions. Hypoxia induced Cygb transcription in heart and liver of both mammals, with the most prominent mRNA up-regulation (12-fold) in Spalax heart. Our data suggest that tissue globins contribute to the remarkable tolerance of Spalax toward environmental hypoxia. This is consistent with the proposed cytoprotective effect of Ngb and Cygb under pathological hypoxic/ischemic conditions in mammals.

Adaptive hypoxic tolerance in the subterranean mole rat Spalax ehrenbergi: the role of vascular endothelial growth factor

Spalax ehrenbergi has evolved adaptations that allow it to survive and carry out normal activities in a highly hypoxic environment. A key component of this adaptation is a higher capillary density in some Spalax tissues resulting in a shorter diffusion distance for oxygen. Vascular endothelial growth factor (VEGF) is an angiogenic factor that is critical for angiogenesis during development and in response to tissue ischemia. We demonstrate here that VEGF expression is markedly increased in those Spalax tissues with a higher capillary density relative to the normal laboratory rat Rattus norvegicus. Upregulation of VEGF thus appears to be an additional mechanism by which Spalax has adapted to its hypoxic environment.

Hypoxia-induced BNIP3 expression and mitophagy: in vivo comparison of the rat and the hypoxia-tolerant mole rat, Spalax ehrenbergi

The blind subterranean mole rat of the Spalax ehrenbergi superspecies is an excellent animal model for hypoxic tolerance. Unique physiological, functional, and gene structure changes allow Spalax species to survive lower oxygen levels than most terrestrial animals. BNIP3, an HIF‐1 dependent hypoxiaresponse gene, has a proapoptotic function;however, expression is suppressed in many types of cancers. Under hypoxic conditions, BNIP3 also functions as a mediator of mitochondrial autophagy, a survival adaptation to control ROS production and DNA damage. Using real‐time PCR and Western blotting, we investigated the impact of hypoxia on BNIP3 expression and mitophagy, in the skeletal muscle and heart, of the Rattus and two Spalax species. BNIP3 transcript, as well as protein levels, increased to significantly higher levels under hypoxia in Rattus tissues, with smaller changes in Spalax. Mitophagy was correlated with BNIP3 expression in the heart with an inverse correlation to hypoxia tolerance. A dense network of vessels in Spalax muscle may offer protection from physiological hypoxia, while the response in Rattus reflects the increase of hypoxic stress. In Spalax tissues, as in many cancers, BNIP3 expression and mitophagy are significantly less affected by hypoxia. Similar mechanisms, beneficial to organisms adapted to stressful environments, may also confer malignant cells with survival features. Understanding the molecular basis of such adaptations may enhance development of new therapeutic modalities.—Band, M., Joel, A., Hernandez, A., Avivi, A. Hypoxiainduced BNIP3 expression and mitophagy: in vivo comparison of the rat and the hypoxia‐tolerant mole rat, Spalax ehrenbergi. FASEB J. 23, 2327–2335 (2009)

Increased blood vessel density provides the mole rat physiological tolerance to its hypoxic subterranean habitat

The blind subterranean mole rat superspecies Spalax ehrenbergi has evolved adaptations that allow it to survive and carry out intensive activities in its highly hypoxic underground sealed burrows. A key component of this adaptation is a higher capillary density in some Spalax tissues, primarily in muscles used in digging and in other energetic activities, resulting in a shorter diffusion distance for oxygen. Vascular endothelial growth factor (VEGF) is an angiogenic factor that is critical for angiogenesis during development and is found in response to tissue ischemia. We demonstrate here that due to physiological differences, the Spalax muscle regulatory mechanism for VEGF is different than in Rattus muscle. In vivo, the constitutive level of the VEGF mRNA and the mRNA levels of its transcriptional regulator HIF‐1α and its mRNA stabilizer HuR are significantly higher in Spalax muscle than in Rattus muscle. Furthermore, as opposed to Rattus, the mRNA levels of HIF‐1α, HuR, VEGF, as well as that of LDH‐A, the enzyme that catalyzes the production of lactate, an accepted marker of anaerobic metabolism, are not increased in Spalax after hypoxia. However, ex vivo, when oxygenation by blood vessels is no longer relevant, the expression pattern of all these genes is similar in the two rodents under both normoxic and hypoxic conditions. Our studies provide evidence that the highly vascularized muscle in Spalax, the most energy consuming tissue during digging, is resistant to the effects of oxygen deprivation. The significance of these results with respect to ischemic vascular disease is abundantly clear.

A Switch from Diurnal to Nocturnal Activity in S. ehrenbergi Is Accompanied by an Uncoupling of Light Input and the Circadian Clock

The subterranean mole rat Spalax ehrenbergi superspecies represents an extreme example of adaptive visual and neuronal reorganization. Despite its total visual blindness, its daily activity rhythm is entrainable to light-dark cycles, indicating that it can confer light information to the clock. Although most individuals are active during the light phase under laboratory conditions (diurnal animals), some individuals switch their activity period to the night (nocturnal animals). Similar to other rodents, the Spalax circadian clock is driven by a set of clock genes, including the period (sPer) genes. In this work, we show that diurnal mole rats express the Per genes sPer1 and sPer2 with a peak during the light period. Light can synchronize sPer gene expression to an altered light-dark cycle and thereby reset the clock. In contrast, nocturnal Spalax express sPer2 in the dark period and sPer1 in a biphasic manner, with a light-dependent maximum during the day and a second light-independent maximum during the night. Although sPer1 expression remains light inducible, this is not sufficient to reset the molecular clockwork. Hence, the strict coupling of light, Per expression, and the circadian clock is lost. This indicates that Spalax can dissociate the light-driven resetting pathway from the central clock oscillator.

Biological clock in total darkness: The Clock/MOP3 circadian system of the blind subterranean mole rat

Blind subterranean mole rats retain a degenerated, subcutaneous, visually blind but functionally circadian eye involved in photoperiodic perception. Here we describe the cloning, sequence, and expression of the circadian Clock and MOP3 cDNAs of the Spalax ehrenbergi superspecies in Israel. Both genes are relatively conserved, although characterized by a significant number of amino acid substitutions. The glutamine-rich area of Clock, which is assumed to function in circadian rhythmicity, is expanded in Spalax compared with that of humans and mice, and is different in amino acid composition from that of rats. We also show that MOP3 is a bona fide partner of Spalax Clock and that the Spalax Clock/MOP3 dimer is less potent than its human counterpart in driving transcription. We suggest that this reduction in transcriptional activity may be attributed to the Spalax Clock glutamine-rich domain, which is unique in its amino acid composition compared with other studied mammalian species. Understanding Clock/MOP3 function could highlight circadian mechanisms in blind mammals and their unique pattern as a result of adapting to life underground.

P53 in blind subterranean mole rats--loss-of-function versus gain-of-function activities on newly cloned Spalax target genes.

A tumor suppressor gene, p53, controls cellular responses to a variety of stress conditions, including DNA damage and hypoxia, leading to growth arrest and/or apoptosis. Recently, we demonstrated that in blind subterranean mole rats, Spalax, a model organism for hypoxia tolerance, the p53 DNA-binding domain contains a specific Arg174Lys amino acid substitution. This substitution reduces the p53 effect on the transcription of apoptosis genes (apaf1, puma, pten and noxa) and enhances it on human cell cycle arrest and p53 stabilization/homeostasis genes (mdm2, pten, p21 and cycG). In the current study, we cloned Spalax apaf1 promoter and mdm2 intronic regions containing consensus p53-responsive elements. We compared the Spalax-responsive elements to those of human, mouse and rat and investigated the transcriptional activity of Spalax and human Arg174Lys-mutated p53 on target genes of both species. Spalax and human-mutated p53 lost induction of apaf1 transcription, and increased induction of mdm2 transcription. We conclude that Spalax evolved hypoxia-adaptive mechanisms, analogous to the alterations acquired by cancer cells during tumor development, with a bias against apoptosis while favoring cell arrest and DNA repair.

Circadian genes in a blind subterranean mammal II: Conservation and uniqueness of the three Period homologs in the blind subterranean mole rat, Spalax ehrenbergi superspecies

We demonstrated that a subterranean, visually blind mammal has a functional set of three Per genes that are important components of the circadian clockwork in mammals. The mole rat superspecies Spalax ehrenbergi is a blind subterranean animal that lives its entire life underground in darkness. It has degenerated eyes, but the retina and highly hypertrophic harderian gland are involved in photoperiodic perception. All three Per genes oscillate with a periodicity of 24 h in the suprachiasmatic nuclei, eye, and harderian gland and are expressed in peripheral organs. This oscillation is maintained under constant conditions. The light inducibility of sPer1 and sPer2, which are similar in structure to those of other mammals, indicates the role of these genes in clock resetting. However, sPer3 is unique in mammals and has two truncated isoforms, and its expressional analysis leaves its function unresolved. Pers expression analysis in the harderian gland suggests an important participation of this organ in the stabilization and resetting mechanism of the central pacemaker in the suprachiasmatic nuclei and in unique adaptation to life underground.

Circadian Genes in a Blind Subterranean Mammal III: Molecular Cloning and Circadian Regulation of Cryptochrome Genes in the Blind Subterranean Mole Rat, Spalax Ehrenbergi Superspecies

The blind subterranean mole rat superspecies Spalax ehrenbergi is an extreme example of mammalian adaptation to life underground. Though this rodent is totally visually blind, harboring a drastically degenerated subcutaneous rudimentary eye, its daily activity rhythm is entrainable to LD cycles. This indicates that it confers light information to the clock, as has been previously shown by the authors in behavioral studies as well as by molecular analyses of its Clock/MOP3 and its three Per genes. The Cryptochrome (Cry) genes found in animals and plants act both as photoreceptors and as essential components of the negative feedback mechanism of the biological clock. To further understand the circadian system of this unique mammal, the authors cloned and characterized the open reading frame of Spalax Cry1 and Cry2. The Spalax CRY1 protein is significantly closer to the human homolog than to the mice one, in contrast to the evolutionary expectations. They have found two isoforms of Cry2 in Spalax, which differ in their 5’ end of the open reading frame and defined their expression in Spalax populations. They found a large and significant excess of heterozygotes of sCry2 (sCry2L/S genotype). Both sCry1 and sCry2 mRNAs were found in the SCN, the eye, the harderian gland, as well as in a wide range of peripheral tissues. Their expression pattern under different LD conditions has also been analyzed. As was already shown for other circadian genes, despite being blind and living in darkness, the Cry genes of Spalax behave in a similar, though not identical, pattern as in sighted animals. Once again, the results indicate that the uniquely hypertrophied harderian gland of Spalax plays a key role in its circadian system.

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.