Low thyroxine serves as an upstream regulator of ecophysiological adaptations in Ansell's mole-rats.

Introduction: About 10% of all rodent species have evolved a subterranean way of life, although life in subterranean burrows is associated with harsh environmental conditions that would be lethal to most animals living above ground. Two key adaptations for survival in subterranean habitats are low resting metabolic rate (RMR) and core body temperature (Tb). However, the upstream regulation of these traits was unknown thus far. Previously, we have reported exceptionally low concentrations of the thyroid hormone (TH) thyroxine (T4), and peculiarities in TH regulating mechanisms in two African mole-rat species, the naked mole-rat and the Ansell's mole-rat. Methods: In the present study, we treated Ansell's mole-rats with T4 for four weeks and analyzed treatment effects on the tissue and whole organism level with focus on metabolism and thermoregulation. Results: We found RMR to be upregulated by T4 treatment but not to the extent that was expected based on serum T4 concentrations. Our data point towards an extraordinary capability of Ansell's mole-rats to effectively downregulate TH signaling at tissue level despite very high serum TH concentrations, which most likely explains the observed effects on RMR. On the other hand, body weight was decreased in T4-treated animals and Tb was upregulated by T4 treatment. Moreover, we found indications of the hypothalamus-pituitary-adrenal axis potentially influencing the treatment effects. Conclusion: Taken together, we provide the first experimental evidence that the low serum T4 concentrations of Ansell's mole-rats serve as an upstream regulator of low RMR and Tb. Thus, our study contributes to a better understanding of the ecophysiological evolution of the subterranean lifestyle in African mole-rats.

Hypoxic and hypercapnic burrow conditions lead to downregulation of free triiodothyronine and hematocrit in Ansell's mole-rats (Fukomys anselli).

African mole-rats live in self-dug burrow systems under hypoxic and hypercapnic conditions. Adaptations to hypoxia include suppression of resting metabolic rate (RMR) and core body temperature (Tb). Because the thyroid hormones (THs) thyroxine (T4) and triiodothyronine (T3) are positive regulators of RMR and Tb, we hypothesized that serum TH concentrations would also be downregulated under hypoxic conditions. To test this hypothesis, we kept Ansell's mole-rats (Fukomys anselli) in terraria filled with soil in which they were allowed to construct underground burrows to achieve chronic intermittent hypoxia and hypercapnia. The animals stayed in these hypoxic and hypercapnic burrows voluntarily, although given the choice to stay aboveground. We collected blood samples before and after treatment to measure serum T4 and T3 concentrations as well as hematological parameters. The free fraction of the transcriptionally-active T3 was significantly decreased after treatment, indicating that cellular TH signaling was downregulated via peripheral mechanisms, consistent with the assumption that aerobic metabolism is downregulated under hypoxic conditions. Furthermore, we found that hematocrit and hemoglobin concentrations were also downregulated after treatment, suggesting that oxygen demand decreases under hypoxia, presumably due to the metabolic shift towards anaerobic metabolism. Taken together, we have identified a potential upstream regulator of physiological adaptations to hypoxia in these highly hypoxia-tolerant animals.

Cigarette smoke causes a bioenergetic crisis in RPE cells involving the downregulation of HIF-1α under normoxia.

Age-related macular degeneration (AMD) is the most common blinding disease in the elderly population. However, there are still many uncertainties regarding the pathophysiology at the molecular level. Currently, impaired energy metabolism in retinal pigment epithelium (RPE) cells is discussed as one major hallmark of early AMD pathophysiology. Hypoxia-inducible factors (HIFs) are important modulators of mitochondrial function. Moreover, smoking is the most important modifiable risk factor for AMD and is known to impair mitochondrial integrity. Therefore, our aim was to establish a cell-based assay that enables us to investigate how smoking affects mitochondrial function in conjunction with HIF signaling in RPE cells. For this purpose, we treated a human RPE cell line with cigarette smoke extract (CSE) under normoxia (21% O2), hypoxia (1% O2), or by co-treatment with Roxadustat, a clinically approved HIF stabilizer. CSE treatment impaired mitochondrial integrity, involving increased mitochondrial reactive oxygen species, disruption of mitochondrial membrane potential, and altered mitochondrial morphology. Treatment effects on cell metabolism were analyzed using a Seahorse Bioanalyzer. Mitochondrial respiration and ATP production were impaired in CSE-treated cells under normoxia. Surprisingly, CSE-treated RPE cells also exhibited decreased glycolytic rate under normoxia, causing a bioenergetic crisis, because two major metabolic pathways that provide ATP were impaired by CSE. Downregulation of glycolytic rate was HIF-dependent because HIF-1α, the α-subunit of HIF-1, was downregulated by CSE on the protein level, especially under normoxia. Moreover, hypoxia incubation and treatment with Roxadustat restored glycolytic flux. Taken together, our in vitro model provides interesting insights into HIF-dependent regulation of glycolysis under normoxic conditions, which will enable us to investigate signaling pathways involved in RPE metabolism in health and disease.

Comparative analysis of thyroid hormone systems in rodents with subterranean lifestyle.

African mole-rats are subterranean rodents inhabiting underground burrows. This habitat entails risks of overheating, hypoxia, and scarce food availability. Consequently, many subterranean species have evolved low basal metabolism and low body temperature, but the regulation of these traits at the molecular level were unknown. Measurements of serum thyroid hormone (TH) concentrations in African mole-rats have revealed a unique TH phenotype, which deviates from the typical mammalian pattern. Since THs are major regulators of metabolic rate and body temperature, we further characterised the TH system of two African mole-rat species, the naked mole-rat (Heterocephalus glaber) and the Ansell's mole-rat (Fukomys anselli) at the molecular level in a comparative approach involving the house mouse (Mus musculus) as a well-studied laboratory model in TH research. Most intriguingly, both mole-rat species had low iodide levels in the thyroid and naked mole-rats showed signs of thyroid gland hyperplasia. However, contrary to expectations, we found several species-specific differences in the TH systems of both mole-rat species, although ultimately resulting in similar serum TH concentrations. These findings indicate a possible convergent adaptation. Thus, our study adds to our knowledge for understanding adaptations to the subterranean habitat.

Macrophage-Orbital Fibroblast Interaction and Hypoxia Promote Inflammation and Adipogenesis in Graves' Orbitopathy.

The inflammatory eye disease Graves' orbitopathy (GO) is the main complication of autoimmune Graves' disease. In previous studies we have shown that hypoxia plays an important role for progression of GO. Hypoxia can maintain inflammation by attracting inflammatory cells such as macrophages (MQ). Herein, we investigated the interaction of MQ and orbital fibroblasts (OF) in context of inflammation and hypoxia. We detected elevated levels of the hypoxia marker HIF-1α, the MQ marker CD68, and inflammatory cytokines TNFα, CCL2, CCL5, and CCL20 in GO biopsies. Hypoxia stimulated GO tissues to release TNFα, CCL2, and CCL20 as measured by multiplex enzyme-linked immunosorbent assay (ELISA). Further, TNFα and hypoxia stimulated the expression of HIF-1α, CCL2, CCL5, and CCL20 in OF derived from GO tissues. Immunofluorescence confirmed that TNFα-positive MQ were present in the GO tissues. Thus, interaction of M1-MQ with OF under hypoxia also induced HIF-1α, CCL2, and CCL20 in OF. Inflammatory inhibitors etanercept or dexamethasone prevented the induction of HIF-1α and release of CCL2 and CCL20. Moreover, co-culture of M1-MQ/OF under hypoxia enhanced adipogenic differentiation and adiponectin secretion. Dexamethasone and HIF-1α inhibitor PX-478 reduced this effect. Our findings indicate that GO fat tissues are characterized by an inflammatory and hypoxic milieu where TNFα-positive MQ are present. Hypoxia and interaction of M1-MQ with OF led to enhanced secretion of chemokines, elevated hypoxic signaling, and adipogenesis. In consequence, M1-MQ/OF interaction results in constant inflammation and tissue remodeling. A combination of anti-inflammatory treatment and HIF-1α reduction could be an effective treatment option.

Hypoxia aggravates ferroptosis in RPE cells by promoting the Fenton reaction.

Oxidative stress and hypoxia in the retinal pigment epithelium (RPE) have long been considered major risk factors in the pathophysiology of age-related macular degeneration (AMD), but systematic investigation of the interplay between these two risk factors was lacking. For this purpose, we treated a human RPE cell line (ARPE-19) with sodium iodate (SI), an oxidative stress agent, together with dimethyloxalylglycine (DMOG) which leads to stabilization of hypoxia-inducible factors (HIFs), key regulators of cellular adaptation to hypoxic conditions. We found that HIF stabilization aggravated oxidative stress-induced cell death by SI and iron-dependent ferroptosis was identified as the main cell death mechanism. Ferroptotic cell death depends on the Fenton reaction where H2O2 and iron react to generate hydroxyl radicals which trigger lipid peroxidation. Our findings clearly provide evidence for superoxide dismutase (SOD) driven H2O2 production fostering the Fenton reaction as indicated by triggered SOD activity upon DMOG + SI treatment as well as by reduced cell death levels upon SOD2 knockdown. In addition, iron transporters involved in non-transferrin-bound Fe2+ import as well as intracellular iron levels were also upregulated. Consequently, chelation of Fe2+ by 2'2-Bipyridyl completely rescued cells. Taken together, we show for the first time that HIF stabilization under oxidative stress conditions aggravates ferroptotic cell death in RPE cells. Thus, our study provides a novel link between hypoxia, oxidative stress and iron metabolism in AMD pathophysiology. Since iron accumulation and altered iron metabolism are characteristic features of AMD retinas and RPE cells, our cell culture model is suitable for high-throughput screening of new treatment approaches against AMD.

Increased longevity due to sexual activity in mole-rats is associated with transcriptional changes in the HPA stress axis.

Sexual activity and/or reproduction are associated with a doubling of life expectancy in the long-lived rodent genus Fukomys. To investigate the molecular mechanisms underlying this phenomenon, we analyzed 636 RNA-seq samples across 15 tissues. This analysis suggests that changes in the regulation of the hypothalamic-pituitary-adrenal stress axis play a key role regarding the extended life expectancy of reproductive vs. non-reproductive mole-rats. This is substantiated by a corpus of independent evidence. In accordance with previous studies, the up-regulation of the proteasome and so-called 'anti-aging molecules', for example, dehydroepiandrosterone, is linked with enhanced lifespan. On the other hand, several of our results are not consistent with knowledge about aging of short-lived model organisms. For example, we found the up-regulation of the insulin-like growth factor 1/growth hormone axis and several other anabolic processes to be compatible with a considerable lifespan prolongation. These contradictions question the extent to which findings from short-lived species can be transferred to longer-lived ones.

Surprisingly long survival of premature conclusions about naked mole-rat biology.

Naked mole-rats express many unusual traits for such a small rodent. Their morphology, social behaviour, physiology, and ageing have been well studied over the past half-century. Many early findings and speculations about this subterranean species persist in the literature, although some have been repeatedly questioned or refuted. While the popularity of this species as a natural-history curiosity, and oversimplified story-telling in science journalism, might have fuelled the perpetuation of such misconceptions, an accurate understanding of their biology is especially important for this new biomedical model organism. We review 28 of these persistent myths about naked mole-rat sensory abilities, ecophysiology, social behaviour, development and ageing, and where possible we explain how these misunderstandings came about.

EyeCi: Optical clearing and imaging of immunolabeled mouse eyes using light-sheet fluorescence microscopy.

Immunofluorescent imaging is an indispensable technique to study morphology and molecular aspects in tissues. Classical approaches make it necessary to cut physical sections of tissue samples to overcome the limited penetration depth of light, restricting the available information to two dimensions. Recent advances in tissue-clearing techniques enable imaging of fluorescently labeled organs and entire organisms on a cellular level in three dimensions without the need of sectioning. Volume imaging of immunolabeled and cleared tissues started a new era of systems biology, because these techniques provide information on connectivity and circuits, especially in structures with projections in three dimensions such as vascular or nervous systems. The variety of published clearing protocols allows the imaging of every organ with a single exception: the eye. Whole-eye clearing approaches were unsuccessful so far due to the strong pigmentation of the retinal pigment epithelium. Here, we present a new protocol that combines a highly effective melanin bleaching step with solvent-based clearing, termed EyeCi. The protocol is compatible with immunolabeling as demonstrated by the visualization of ocular and retinal vasculature in the intact mouse eye by means of light-sheet fluorescence microscopy. This novel protocol is rapid (1 week) and inexpensive, hence allowing high-throughput, high resolution analysis of vascular architecture of healthy and diseased eyes, in its native, three-dimensional organization within intact eyeballs. Volume imaging of whole cleared eyeballs further enables three-dimensional surface reconstruction and automated quantification of choroidal and retinal vasculature extending ocular imaging to a global level. Thus, EyeCi represents an extension to state-of-the-art light microscopy techniques and is potentially suitable for the investigation of vascular leakage or neovascularization processes.

Higher gene expression stability during aging in long-lived giant mole-rats than in short-lived rats.

Many aging-associated physiological changes are known to occur in short- and long-lived species with different trajectories. Emerging evidence suggests that numerous life history trait differences between species are based on interspecies variations in gene expression. Little information is available, however, about differences in transcriptome changes during aging between mammals with diverging lifespans. For this reason, we studied the transcriptomes of five tissue types and two age cohorts of two similarly sized rodent species with very different lifespans: laboratory rats (Rattus norvegicus) and giant mole-rats (Fukomys mechowii), with maximum lifespans of 3.8 and more than 20 years, respectively. Our findings show that giant mole-rats exhibit higher gene expression stability during aging than rats. Although well-known aging signatures were detected in all tissue types of rats, they were found in only one tissue type of giant mole-rats. Furthermore, many differentially expressed genes that were found in both species were regulated in opposite directions during aging. This suggests that expression changes which cause aging in short-lived species are counteracted in long-lived species. Taken together, we conclude that expression stability in giant mole rats (and potentially in African mole-rats in general) may be one key factor for their long and healthy life.

Retinal S-opsin dominance in Ansell's mole-rats (Fukomys anselli) is a consequence of naturally low serum thyroxine.

Mammals usually possess a majority of medium-wavelength sensitive (M-) and a minority of short-wavelength sensitive (S-) opsins in the retina, enabling dichromatic vision. Unexpectedly, subterranean rodents from the genus Fukomys exhibit an S-opsin majority, which is exceptional among mammals, albeit with no apparent adaptive value. Because thyroid hormones (THs) are pivotal for M-opsin expression and metabolic rate regulation, we have, for the first time, manipulated TH levels in the Ansell's mole-rat (Fukomys anselli) using osmotic pumps. In Ansell's mole-rats, the TH thyroxine (T4) is naturally low, likely as an adaptation to the harsh subterranean ecological conditions by keeping resting metabolic rate (RMR) low. We measured gene expression levels in the eye, RMR, and body mass (BM) in TH-treated animals. T4 treatment increased both, S- and M-opsin expression, albeit M-opsin expression at a higher degree. However, this plasticity was only given in animals up to approximately 2.5 years. Mass-specific RMR was not affected following T4 treatment, although BM decreased. Furthermore, the T4 inactivation rate is naturally higher in F. anselli compared to laboratory rodents. This is the first experimental evidence that the S-opsin majority in Ansell's mole-rats is a side effect of low T4, which is downregulated to keep RMR low.

Higher resting metabolic rate in long-lived breeding Ansell's mole-rats (Fukomys anselli).

BACKGROUND: Reproduction is an energetically expensive process that supposedly impairs somatic integrity in the long term, because resources are limited and have to be allocated between reproduction and somatic maintenance, as predicted by the life history trade-off model. The consequence of reduced investment in somatic maintenance is a gradual deterioration of function, i.e. senescence. However, this classical trade-off model gets challenged by an increasing number of contradicting studies. Here we report about an animal model, which adds more complexity to the ongoing debate. Ansell's mole-rats are long-lived social subterranean rodents with only the founder pair reproducing, while most of their offspring remain in the parental burrow system and do not breed. Despite of a clear reproductive trade-off, breeders live up to twice as long as non-breeders, a unique feature amongst mammals. METHODS: We investigated mass-specific resting metabolic rates (msRMR) of breeders and non-breeders to gain information about the physiological basis underlying the reproduction-associated longevity in Ansell's mole-rats. We assessed the thermoneutral zone (TNZ) for breeders and non-breeders separately by means of indirect calorimetry. We applied generalized linear mixed-effects models for repeated measurements using the msRMR in the respective TNZs. RESULTS: TNZ differed between reproductive and non-reproductive Ansell's mole-rats. Contrary to classical aging models, the shorter-lived non-breeders had significantly lower msRMR within the thermoneutral zone compared to breeders. CONCLUSION: This is the first study reporting a positive correlation between msRMR and lifespan based on reproductive status. Our finding contradicts common aging theories, but supports recently introduced models which do not necessarily link reproductive trade-offs to lifespan reduction.

Audiograms of three subterranean rodent species (genus Fukomys) determined by auditory brainstem responses reveal extremely poor high-frequency hearing.

Life underground has shaped the auditory sense of subterranean mammals, shifting their hearing range to low frequencies. Mole-rats of the genus Fukomys have, however, been suggested to hear at frequencies up to 18.5 kHz, unusually high for a subterranean rodent. We present audiograms of three mole-rat species, Fukomys anselli, Fukomys micklemi and the giant mole-rat Fukomys mechowii, based on evoked auditory brainstem potentials. All species showed low sensitivity and restricted hearing ranges at 60 dB SPL extending from 125 Hz to 4 kHz (5 octaves) with most-sensitive hearing between 0.8 kHz and 1.4 kHz. The high-frequency cut-offs are the lowest found in mammals to date. In contrast to predictions from middle ear morphology, F. mechowii did not show higher sensitivity than F. anselli in the low-frequency range. These data suggest that the hearing range of Fukomys mole-rats is highly restricted to low frequencies and similar to that of other subterranean mammals.

Age-Dependent Changes of Monocarboxylate Transporter 8 Availability in the Postnatal Murine Retina.

The thyroid hormones (TH) triiodothyronine (T3) and its prohormone thyroxine (T4) are crucial for retinal development and function, and increasing evidence points at TH dysregulation as a cause for retinal degenerative diseases. Thus, precise regulation of retinal TH supply is required for proper retinal function, but knowledge on these mechanisms is still fragmentary. Several transmembrane transporters have been described as key regulators of TH availability in target tissues of which the monocarboxylate transporter 8 (MCT8), a high affinity transporter for T4 and T3, plays an essential role in the central nervous system. Moreover, in the embryonic chicken retina, MCT8 is highly expressed, but the postnatal availability of MCT8 in the mammalian retina was not reported to date. In the present study, spatiotemporal retinal MCT8 availability was examined in mice of different age. For this purpose, we quantified expression levels of Mct8 via Real-Time Reverse-Transcriptase PCR in mouse eyecups (C57BL/6) of juvenile and adult age groups. Additionally, age-dependent MCT8 protein levels were quantified via Western blotting and localized via immunofluorescence confocal microscopy. While no difference in Mct8 expression levels could be detected between age groups, MCT8 protein levels in juvenile animals were about two times higher than in adult animals based on Western blot analyses. Immunohistochemical analyses showed that MCT8 immunoreactivity in the eyecup was restricted to the retina and the retinal pigment epithelium. In juvenile mice, MCT8 was broadly observed along the apical membrane of the retinal pigment epithelium, tightly surrounding photoreceptor outer segments. Distinct immunopositive staining was also detected in the inner nuclear layer and the ganglion cell layer. However, in adult specimens, immunoreactivity visibly declined in all layers, which was in line with Western blot analyses. Since MCT8 was abundantly present in juvenile and about twofold lower in adult retinae, our findings suggest a pivotal role of MCT8 especially during postnatal maturation. The present study provides novel insights into age-dependent retinal TH supply, which might help to understand different aspects regarding retinal development, function, and disorders.

Temperature preferences of African mole-rats (family Bathyergidae).

Many animals are able to detect small temperature differences and show strong temperature preferences during periods of rest and activity. Mammals inhabiting the subterranean ecotope can adapt their digging and foraging activity in shallow tunnels temporarily to periods with favourable ambient air and soil temperatures. Moreover, subterranean mammals have the unique opportunity to select for their nests in soil depths with certain, daily and seasonally constant temperatures. Our knowledge on nest temperatures in several species of subterranean mammals is based on measurements of temperatures in empty nests. We can expect, however, that the temperature in an occupied nest is higher (due to the "igloo effect"). We performed two experiments regarding the temperature preference in five species of African mole-rats (Bathyergidae, Rodentia: Fukomys anselli, F. mechowii, F. micklemi, Heliophobius argenteocinereus, and Heterocephalus glaber). In a first experiment, the animals were tested pairwise (except for the solitary silvery mole-rats, H. argenteocinereus, that were tested singly) in an apparatus consisting of seven chambers with a temperature gradient ranging between 16 and 37°C (air temperature). While the smaller species (<110g; F. anselli, F. micklemi, H. glaber) chose chambers with average air temperatures around 29°C, the larger mole-rats rested preferably at lower temperatures of approximately 25.6°C (F. mechowii) and 27.7°C (H. argenteocinereus). A strong negative correlation between body mass and preferred air temperature was detected across species. Thus, the results comply with the surface-volume-rule. Contrary to expectations, temperature preference of naked mole-rats (H. glaber) did not deviate from those of furred small mole-rats, but followed the general trend with smaller species preferring higher temperatures. In a second experiment, Ansell's mole-rats (F. anselli) were tested in groups of four, six and nine animals and the preferred temperatures were compared to the values obtained for pairs. The preferred mean air temperatures did not differ among the groups of different sizes. We discuss our findings in the light of ecophysiological adaptations to cope with the ambient conditions proposed by the "thermal stress hypothesis". Furthermore, we suggest that while soil temperature is decisive during digging as the mole-rats warm up or cool due to tight contact between body and soil (conduction), resting animals prevent heat loss through conduction by building a nest.

Unusual ratio between free thyroxine and free triiodothyronine in a long-lived mole-rat species with bimodal ageing.

Ansell's mole-rats (Fukomys anselli) are subterranean, long-lived rodents, which live in eusocial families, where the maximum lifespan of breeders is twice as long as that of non-breeders. Their metabolic rate is significantly lower than expected based on allometry, and their retinae show a high density of S-cone opsins. Both features may indicate naturally low thyroid hormone levels. In the present study, we sequenced several major components of the thyroid hormone pathways and analyzed free and total thyroxine and triiodothyronine in serum samples of breeding and non-breeding F. anselli to examine whether a) their thyroid hormone system shows any peculiarities on the genetic level, b) these animals have lower hormone levels compared to euthyroid rodents (rats and guinea pigs), and c) reproductive status, lifespan and free hormone levels are correlated. Genetic analyses confirmed that Ansell's mole-rats have a conserved thyroid hormone system as known from other mammalian species. Interspecific comparisons revealed that free thyroxine levels of F. anselli were about ten times lower than of guinea pigs and rats, whereas the free triiodothyronine levels, the main biologically active form, did not differ significantly amongst species. The resulting fT4:fT3 ratio is unusual for a mammal and potentially represents a case of natural hypothyroxinemia. Comparisons with total thyroxine levels suggest that mole-rats seem to possess two distinct mechanisms that work hand in hand to downregulate fT4 levels reliably. We could not find any correlation between free hormone levels and reproductive status, gender or weight. Free thyroxine may slightly increase with age, based on sub-significant evidence. Hence, thyroid hormones do not seem to explain the different ageing rates of breeders and non-breeders. Further research is required to investigate the regulatory mechanisms responsible for the unusual proportion of free thyroxine and free triiodothyronine.