African mole-rats provide a unique taxonomic group for investigating the evolution and neurobiology of sociality. The two species investigated here display extreme differences in social organization and reproductive strategy. Naked mole-rats (NMRs) live in colonies, dominated by a queen and her consorts; most members remain nonreproductive throughout life but cooperate in burrowing, foraging, and caring for pups, for which they are not biological parents (alloparenting). In contrast, Cape mole-rats (CMRs) are solitary and intolerant of conspecifics, except during fleeting seasonal copulation or minimal maternal behavior. Research on other mammals suggests that oxytocin receptors at various telencephalic sites regulate social recognition, monogamous pair bonding, and maternal/allomaternal behavior. Current paradigms in this field derive from monogamous and polygamous species of New World voles, which are evolutionarily remote from Old World mole-rats. The present findings indicate that NMRs exhibit a considerably greater level of oxytocin receptor (OTR) binding than CMRs in the: nucleus accumbens; indusium griseum; central, medial, and cortical amygdaloid nuclei; bed nucleus of the stria terminalis; and CA1 hippocampal subfield. In contrast, OTR binding in the piriform cortex is intense in CMRs but undetectable in NMRs. We speculate that the abundance of OTR binding and oxytocin-neurophysin-immunoreactive processes in the nucleus accumbens of NMRs reflects their sociality, alloparenting behavior, and potential for reproductive attachments. In contrast, the paucity of oxytocin and its receptors at this site in CMRs may reflect a paucity of prosocial behaviors. Whether similarities in OTR expression between eusocial mole-rats and monogamous voles are due to gene conservation or convergent evolution remains to be determined.

Life underground often leads to animals having specialized auditory systems to accommodate the constraints of acoustic transmission in tunnels. Despite living underground, naked mole-rats use a highly vocal communication system, implying that they rely on central auditory processing. However, little is known about these animals' central auditory system, and whether it follows a similar developmental time course as other rodents. Naked mole-rats show slowed development in the hippocampus suggesting they have altered brain development compared to other rodents. Here, we measured morphological characteristics and voltage-gated potassium channel Kv3.3 expression and protein levels at different key developmental time points (postnatal days 9, 14, 21 and adulthood) to determine whether the auditory brainstem (lateral superior olive and medial nucleus of the trapezoid body) develops similarly to two common auditory rodent model species: gerbils and mice. Additionally, we measured the hearing onset of naked mole-rats using auditory brainstem response recordings at the same developmental timepoints. In contrast with other work in naked mole-rats showing that they are highly divergent in many aspects of their physiology, we show that naked mole-rats have a similar hearing onset, between postnatal day (P) 9 and P14, to many other rodents. On the other hand, we show some developmental differences, such as a unique morphology and Kv3.3 protein levels in the brainstem.

Osteoarthritis (OA) is the most prevalent disabling disease, affecting quality of life and contributing to morbidity, particularly during aging. Current treatments for OA are limited to palliation: pain management and surgery for end-stage disease. Innovative approaches and animal models are needed to develop curative treatments for OA. Here, we investigated the naked mole-rat (NMR) as a potential model of OA resistance. NMR is a small rodent with the maximum lifespan of over 30 years, resistant to a wide range of age-related diseases. NMR tissues accumulate large quantities of unique, very high molecular weight, hyaluronan (HA). HA is a major component of cartilage and synovial fluid. Importantly, both HA molecular weight and cartilage stiffness decline with age and progression of OA. As increased polymer length is known to result in stiffer material, we hypothesized that NMR high molecular weight HA contributes to stiffer cartilage. Our analysis of biomechanical properties of NMR cartilage revealed that it is significantly stiffer than mouse cartilage. Furthermore, NMR chondrocytes were highly resistant to traumatic damage. In vivo experiments using an injury-induced model of OA revealed that NMRs were highly resistant to OA. While similarly treated mice developed severe cartilage degeneration, NMRs did not show any signs of OA. Our study shows that NMRs are remarkably resistant to OA, and this resistance is likely conferred by high molecular weight HA. This work suggests that NMR is a useful model to study OA resistance and NMR high molecular weight HA may hold therapeutic potential for OA treatment.

The Olfactory Receptor (OR) superfamily, the largest in the vertebrate genome, is responsible for vertebrate olfaction and is traditionally subdivided into 17 OR families. Recent studies characterising whole-OR subgenomes revealed a 'birth and death' model of evolution for a range of species, however little is known about fine-scale evolutionary dynamics within single-OR families. This study reports the first assessment of fine-scale OR evolution and variation in African mole-rats (Bathyergidae), a family of subterranean rodents endemic to sub-Saharan Africa. Because of the selective pressures of life underground, enhanced olfaction is proposed to be fundamental to the evolutionary success of the Bathyergidae, resulting in a highly diversified OR gene-repertoire. Using a PCR-sequencing approach, we analysed variation in the OR7 family across 14 extant bathyergid species, which revealed enhanced levels of functional polymorphisms concentrated across the receptors' ligand-binding region. We propose that mole-rats are able to recognise a broad range of odorants and that this diversity is reflected throughout their OR7 gene repertoire. Using both classic tests and tree-based methods to test for signals of selection, we investigate evolutionary forces across the mole-rat OR7 gene tree. Four well-supported clades emerged in the OR phylogeny, with varying signals of selection; from neutrality to positive and purifying selection. Bathyergid life-history traits and environmental niche-specialisation are explored as possible drivers of adaptive OR evolution, emerging as non-exclusive contributors to the positive selection observed at OR7 genes. Our results reveal unexpected complexity of evolutionary mechanisms acting within a single OR family, providing insightful perspectives into OR evolutionary dynamics.

The concept of climate variability facilitating adaptive radiation supported by the "Court Jester" hypothesis is disputed by the "Red Queen" one, but the prevalence of one or the other might be scale-dependent. We report on a detailed, comprehensive phylo-geographic study on the ∼4 kb mtDNA sequence in underground blind mole rats of the family Spalacidae (or subfamily Spalacinae) from the East Mediterranean steppes. Our study aimed at testing the presence of periodicities in branching patterns on a constructed phylogenetic tree and at searching for congruence between branching events, tectonic history and paleoclimates. In contrast to the strong support for the majority of the branching events on the tree, the absence of support in a few instances indicates that network-like evolution could exist in spalacids. In our tree, robust support was given, in concordance with paleontological data, for the separation of spalacids from muroid rodents during the first half of the Miocene when open, grass-dominated habitats were established. Marine barriers formed between Anatolia and the Balkans could have facilitated the separation of the lineage "Spalax" from the lineage "Nannospalax" and of the clade "leucodon" from the clade "xanthodon". The separation of the clade "ehrenbergi" occurred during the late stages of the tectonically induced uplift of the Anatolian high plateaus and mountains, whereas the separation of the clade "vasvarii" took place when the rapidly uplifting Taurus mountain range prevented the Mediterranean rainfalls from reaching the Central Anatolian Plateau. The separation of Spalax antiquus and S. graecus occurred when the southeastern Carpathians were uplifted. Despite the role played by tectonic events, branching events that show periodicity corresponding to 400-kyr and 100-kyr eccentricity bands illuminate the important role of orbital fluctuations on adaptive radiation in spalacids. At the given scale, our results supports the "Court Jester" hypothesis over the "Red Queen" one.

Neuropathic pain is a chronic disease state resulting from injury to the nervous system. This type of pain often responds poorly to standard treatments and occasionally may get worse instead of better over time. Patients who experience neuropathic pain report sensitivity to cold and mechanical stimuli. Since the nociceptive system of African naked mole-rats contains unique adaptations that result in insensitivity to some pain types, we investigated whether naked mole-rats may be resilient to sensitivity following nerve injury. Using the spared nerve injury model of neuropathic pain, we showed that sensitivity to mechanical stimuli developed similarly in mice and naked mole-rats. However, naked mole-rats lacked sensitivity to mild cold stimulation after nerve injury, while mice developed robust cold sensitivity. We pursued this response deficit by testing behavior to activators of transient receptor potential (TRP) receptors involved in detecting cold in naïve animals. Following mustard oil, a TRPA1 activator, naked mole-rats responded similarly to mice. Conversely, icilin, a TRPM8 agonist, did not evoke pain behavior in naked mole-rats when compared with mice. Finally, we used RNAscope to probe for TRPA1 and TRPM8 messenger RNA expression in dorsal root ganglia of both species. We found increased TRPA1 messenger RNA, but decreased TRPM8 punctae in naked mole-rats when compared with mice. Our findings likely reflect species differences due to evolutionary environmental responses that are not easily explained by differences in receptor expression between the species.

The naked mole-rat (Heterocephalus glaber) is characterized by a more than tenfold higher life expectancy compared to another rodent species of the same size, namely, the laboratory mouse (Mus musculus). We used mass spectrometric metabolomics to analyze circulating plasma metabolites in both species at different ages. Interspecies differences were much more pronounced than age-associated alterations in the metabolome. Such interspecies divergences affected multiple metabolic pathways involving amino, bile and fatty acids as well as monosaccharides and nucleotides. The most intriguing metabolites were those that had previously been linked to pro-health and antiaging effects in mice and that were significantly increased in the long-lived rodent compared to its short-lived counterpart. This pattern applies to α-tocopherol (also known as vitamin E) and polyamines (in particular cadaverine, N8-acetylspermidine and N1,N8-diacetylspermidine), all of which were more abundant in naked mole-rats than in mice. Moreover, the age-associated decline in spermidine and N1-acetylspermidine levels observed in mice did not occur, or is even reversed (in the case of N1-acetylspermidine) in naked mole-rats. In short, the present metabolomics analysis provides a series of testable hypotheses to explain the exceptional longevity of naked mole-rats.

Compared to many other rodent species, naked mole rats (Heterocephalus glaber) have elevated auditory thresholds, poor frequency selectivity, and limited ability to localize sound. Because the cochlea is responsible for encoding and relaying auditory signals to the brain, we used immunofluorescence and quantitative image analysis to examine cochlear innervation in mature and developing naked mole rats compared to mice (Mus musculus), gerbils (Meriones unguiculatus), and Damaraland mole rats (Fukomys damarensis), another subterranean rodent. In comparison to mice and gerbils, we observed alterations in afferent and efferent innervation as well as their patterns of developmental refinement in naked and Damaraland mole rats. These alterations were, however, not always shared similarly between naked and Damaraland mole rats. Most conspicuously, in both naked and Damaraland mole rats, inner hair cell (IHC) afferent ribbon density was reduced, whereas outer hair cell afferent ribbon density was increased. Naked and Damaraland mole rats also showed reduced lateral and medial efferent terminal density. Developmentally, naked mole rats showed reduced and prolonged postnatal reorganization of afferent and efferent innervation. Damaraland mole rats showed no evidence of postnatal reorganization. Differences in cochlear innervation specifically between the two subterranean rodents and more broadly among rodents provides insight into the cochlear mechanisms that enhance frequency sensitivity and sound localization, maturation of the auditory system, and the evolutionary adaptations occurring in response to subterranean environments.

The domestic and foreign scholars have studied naked mole rats more focused on the respect such as its long life, resistant to low oxygen, little spontaneous tumor, but the study of the immune system is little. In this study, we compared the anatomy and tissue morphology of NMR and ICR mouse spleens and found that the gross appearance of the NNMR spleen differed from ICR. There were more macrophages in NNMR spleens than in ICR spleens. Furthermore, we focused on the differences of macrophages. We compared their phagocytic capabilities and the data showed that NNMR macrophages are more phagocytic than ICR mouse macrophages. We also used polyI:C and LPS to stimulate the NMR and ICR macrophages and then measured the immune response as expression of certain TLR signaling molecules. After stimulation, there was a lower increase in apoptosis of NMR macrophages than ICR macrophages and a non-significant increased expression of TLRs in NMR macrophages than in ICR macrophages. In contrast, NF-κB proteins increased more significantly in NMR's than in ICR's and the expression of downstream cytokines in NMR macrophages also increased more than in ICR macrophages. Based on these results, we hypothesize that in addition to being able to eat foreign matter, NMR macrophages can activate the TLRs, start the NF-κB and produce a large number of cytokines to enhance immune response, so as to protect the body from outside interference when the virus or bacteria invading.

The long-living naked mole-rat (NMR) shows negligible senescence and resistance to age-associated diseases. Recent evidence, based on protein-level assays, suggests that enhanced protein homeostasis machinery contributes to NMR stress-resistance and longevity. Here, we develop NMR-specific, transcriptional assays for measuring the unfolded protein response (UPR), a component of ER proteostasis. By varying doses and response times of pharmacological ER stressors applied to NMR kidney fibroblasts, we probe the NMR UPR in detail, demonstrating that NMR fibroblasts have a higher UPR activation threshold compared to mouse fibroblasts under mild ER-stress induction; whereas temporal analysis reveals that severe ER-stress induction results in no comparative differences. Probing NMR UPR activation with our robust assays may lead to insights into the proteostasis and ageing relationship.

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.

Immunosenescence is a hallmark of aging and manifests as increased susceptibility to infection, autoimmunity, and cancer in the elderly. One component of immunosenescence is thymic involution, age-associated shrinkage of the thymus, observed in all vertebrates studied to date. The naked mole rat (Heterocephalus glaber) has become an attractive animal model in aging research due to its extreme longevity and resistance to disease. Here, we show that naked mole rats display no thymic involution up to 11 years of age. Furthermore, we found large ectopic cervical thymi in addition to the canonical thoracic thymus, both being identical in their cell composition. The developmental landscape in naked mole rat thymi revealed overt differences from the murine T-cell compartment, most notably a decrease of CD4+ /CD8+ double-positive cells and lower abundance of cytotoxic effector T cells. Our observations suggest that naked mole rats display a delayed immunosenescence. Therapeutic interventions aimed at reversing thymic aging remain limited, underscoring the importance of understanding the cellular and molecular mechanisms behind a sustained immune function in the naked mole rat.

Subterranean common mole-rats of the genus Fukomys (family Bathyergidae) live in large, cooperatively-breeding families. Odor cues have been hypothesized to play an important role in mediating social behaviors in the underground ecotope, but only little is known about the role of olfactory signaling in burrowing mammals. Here we characterize the so far neglected perioral glands of Fukomys and other African mole-rats as an important source of olfactory social information. Histology demonstrates these structures to be derived sebaceous glands that are developed regardless of sex and reproductive status. However, gland activity is higher in Fukomys males, leading to sexually dimorphic patterns of stain and clotting of the facial pelage. Behavioral assays revealed that conspecifics prefer male but not female perioral swabs over scent samples from the back fur and that male sebum causes similar attraction as anogenital scent, a known source of social information in Fukomys. Finally, we assessed volatile compounds in the perioral sebum of the giant mole-rat (Fukomys mechowii) via GCxGC-MS-based metabolomic profiling. Volatiles display pronounced sex-specific signatures but also allow to differentiate between intrasexual reproductive status groups. These different lines of evidence suggest that mole-rat perioral glands provide complex odor signals which play a crucial role in social communication.

Naked mole-rats are highly vocal, eusocial, subterranean rodents with, counterintuitively, poor hearing. The causes underlying their altered hearing are unknown. Moreover, whether altered hearing is degenerate or adaptive to their unique lifestyles is controversial. We used various methods to identify the factors contributing to altered hearing in naked and the related Damaraland mole-rats and to examine whether these alterations result from relaxed or adaptive selection. Remarkably, we found that cochlear amplification was absent from both species despite normal prestin function in outer hair cells isolated from naked mole-rats. Instead, loss of cochlear amplification appears to result from abnormal hair bundle morphologies observed in both species. By exploiting a well-curated deafness phenotype-genotype database, we identified amino acid substitutions consistent with abnormal hair bundle morphology and reduced hearing sensitivity. Amino acid substitutions were found in unique groups of six hair bundle link proteins. Molecular evolutionary analyses revealed shifts in selection pressure at both the gene and the codon level for five of these six hair bundle link proteins. Substitutions in three of these proteins are associated exclusively with altered hearing. Altogether, our findings identify the likely mechanism of altered hearing in African mole-rats, making them the only identified mammals naturally lacking cochlear amplification. Moreover, our findings suggest that altered hearing in African mole-rats is adaptive, perhaps tailoring hearing to eusocial and subterranean lifestyles. Finally, our work reveals multiple, unique evolutionary trajectories in African mole-rat hearing and establishes species members as naturally occurring disease models to investigate human hearing loss.

The naked mole-rat (NMR, Heterocephalus glaber), which is the longest-lived rodent species, exhibits extraordinary resistance to cancer. Here we report that NMR somatic cells exhibit a unique tumour-suppressor response to reprogramming induction. In this study, we generate NMR-induced pluripotent stem cells (NMR-iPSCs) and find that NMR-iPSCs do not exhibit teratoma-forming tumorigenicity due to the species-specific activation of tumour-suppressor alternative reading frame (ARF) and a disruption mutation of the oncogene ES cell-expressed Ras (ERAS). The forced expression of Arf in mouse iPSCs markedly reduces tumorigenicity. Furthermore, we identify an NMR-specific tumour-suppression phenotype-ARF suppression-induced senescence (ASIS)-that may protect iPSCs and somatic cells from ARF suppression and, as a consequence, tumorigenicity. Thus, NMR-specific ARF regulation and the disruption of ERAS regulate tumour resistance in NMR-iPSCs. Our findings obtained from studies of NMR-iPSCs provide new insight into the mechanisms of tumorigenicity in iPSCs and cancer resistance in the NMR.

The naked mole rat (NMR), Heterocephalus glaber, the longest-living rodent, provides a unique opportunity to explore how evolution has shaped adult stem cell (ASC) activity and tissue function with increasing lifespan. Using cumulative BrdU labelling and a quantitative imaging approach to track intestinal ASCs (Lgr5+) in their native in vivo state, we find an expanded pool of Lgr5+ cells in NMRs, and these cells specifically at the crypt base (Lgr5+CBC) exhibit slower division rates compared to those in short-lived mice but have a similar turnover as human LGR5+CBC cells. Instead of entering quiescence (G0), NMR Lgr5+CBC cells reduce their division rates by prolonging arrest in the G1 and/or G2 phases of the cell cycle. Moreover, we also observe a higher proportion of differentiated cells in NMRs that confer enhanced protection and function to the intestinal mucosa which is able to detect any chemical imbalance in the luminal environment efficiently, triggering a robust pro-apoptotic, anti-proliferative response within the stem/progenitor cell zone.

Naked mole rats (NMRs) exhibit blunted hypoxic (HVR) and hypercapnic ventilatory responses (HCVR). The mechanism(s) underlying these responses are largely unknown. We hypothesized that attenuated carotid body (CB) sensitivity to hypoxia and hypercapnia contributes to the near absence of ventilatory responses to hypoxia and CO2 in NMRs.

In the long-lived naked mole-rat (NMR), the entire process of oogenesis occurs postnatally. Germ cell numbers increase significantly in NMRs between postnatal days 5 (P5) and P8, and germs cells positive for proliferation markers (Ki-67, pHH3) are present at least until P90. Using pluripotency markers (SOX2 and OCT4) and the primordial germ cell (PGC) marker BLIMP1, we show that PGCs persist up to P90 alongside germ cells in all stages of female differentiation and undergo mitosis both in vivo and in vitro. We identified VASA+ SOX2+ cells at 6 months and at 3-years in subordinate and reproductively activated females. Reproductive activation was associated with proliferation of VASA+ SOX2+ cells. Collectively, our results suggest that highly desynchronized germ cell development and the maintenance of a small population of PGCs that can expand upon reproductive activation are unique strategies that could help to maintain the NMR's ovarian reserve for its 30-year reproductive lifespan.

Fertility is abolished in nonbreeding males in colonies of natal naked mole-rats (NMRs). Although spermatogenesis occurs in both breeding and nonbreeding male NMRs, the mechanisms underlying the differences in fertility between breeders and nonbreeders remain unexplored. In this study, a significant decrease in autophagy was observed in Leydig cells of the testis from nonbreeding male NMRs. This alteration was visualised as a significant decrease in the levels of autophagy-related gene 7 (Atg7), Atg5, microtubule-associated protein 1A/B light chain 3 (LC3-II/I) and the number of autophagosomes and an increase in P62 levels using Western blotting analyses. Furthermore, monodansylcadaverine (MDC) staining and Western blot analyses revealed that testosterone production decreased in nonbreeding male NMR Leydig cells, this decrease was associated with a reduction in autophagy. Primary Leydig cells from breeding and nonbreeding male NMRs were processed to investigate the effect of an autophagy inhibitor (3-MA, 3-methyladenine) or an autophagy activator (rapamycin) on testosterone production. Rapamycin induced an increase in testosterone production in NMR Leydig cells, whereas 3-MA had the opposite effect. Consequently, spermatogenesis, the weight of the testis, and androgen levels were dramatically reduced in nonbreeding male NMRs. While rapamycin treatment restored the fertility of nonbreeding male NMRs. Based on these results, inadequate autophagy correlates with a decrease in steroid production in nonbreeding male NMR Leydig cells, which may ultimately influence the spermatogenesis and fertilities of these animals.

The social brain hypothesis (SBH) posits that the demands imposed on individuals by living in cohesive social groups exert a selection pressure favouring the evolution of large brains and complex cognitive abilities. Using volumetry and the isotropic fractionator to determine the size of and numbers of neurons in specific brain regions, here we test this hypothesis in African mole-rats (Bathyergidae). These subterranean rodents exhibit a broad spectrum of social complexity, ranging from strictly solitary through to eusocial cooperative breeders, but feature similar ecologies and life history traits. We found no positive association between sociality and neuroanatomical correlates of information-processing capacity. Solitary species are larger, tend to have greater absolute brain size and have more neurons in the forebrain than social species. The neocortex ratio and neuronal counts correlate negatively with social group size. These results are clearly inconsistent with the SBH and show that the challenges coupled with sociality in this group of rodents do not require brain enlargement or fundamental reorganization. These findings suggest that group living or pair bonding per se does not select strongly for brain enlargement unless coupled with Machiavellian interactions affecting individual fitness.