Dysregulation of ADAM10 shedding activity in naked mole-rat fibroblasts is due to deficient phosphatidylserine externalization.

The naked mole-rat (NMR, Heterocephalus glaber) is of significant interest to biogerontological research, rarely developing age-associated diseases, such as cancer. The transmembrane glycoprotein CD44 is upregulated in certain cancers and CD44 cleavage by a disintegrin and metalloproteinase 10 (ADAM10) regulates cellular migration. Here we provide evidence that mature ADAM10 is expressed in NMR primary skin fibroblasts (NPSF), and that ionomycin increases cell surface ADAM10 localization. However, we observed an absence of ADAM10 mediated CD44 cleavage, as well as shedding of exogenous and overexpressed betacellulin in NPSF, whereas in mouse primary skin fibroblasts ionomycin induced ADAM10-dependent cleavage of both CD44 and betacellulin. Overexpressing a hyperactive form of the Ca2+ -dependent phospholipid scramblase ANO6 in NPSF increased phosphatidylserine (PS) externalization, which rescued the ADAM10 sheddase activity and promoted cell migration in NPSF in an ADAM10-dependent manner. These findings suggest that dysregulation of ADAM10 shedding activity is due to a deficient PS externalization in NMR.

Sensitization of colonic nociceptors by IL-13 is dependent on JAK and p38 MAPK activity.

The effective management of visceral pain is a significant unmet clinical need for those affected by gastrointestinal diseases, such as inflammatory bowel disease (IBD). The rational design of novel analgesics requires a greater understanding of the mediators and mechanisms underpinning visceral pain. Interleukin-13 (IL-13) production by immune cells residing in the gut is elevated in IBD, and IL-13 appears to be important in the development of experimental colitis. Furthermore, receptors for IL-13 are expressed by neurons innervating the colon, though it is not known whether IL-13 plays any role in visceral nociception per se. To resolve this, we used Ca2+ imaging of cultured sensory neurons and ex vivo electrophysiological recording from the lumbar splanchnic nerve innervating the distal colon. Ca2+ imaging revealed the stimulation of small-diameter, capsaicin-sensitive sensory neurons by IL-13, indicating that IL-13 likely stimulates nociceptors. IL-13-evoked Ca2+ signals were attenuated by inhibition of Janus (JAK) and p38 kinases. In the lumbar splanchnic nerve, IL-13 did not elevate baseline firing, nor sensitize the response to capsaicin application, but did enhance the response to distention of the colon. In line with Ca2+ imaging experiments, IL-13-mediated sensitization of the afferent response to colon distention was blocked by inhibition of either JAK or p38 kinase signaling. Together, these data highlight a potential role for IL-13 in visceral nociception and implicate JAK and p38 kinases in pronociceptive signaling downstream of IL-13.

Sensitization of colonic nociceptors by TNFα is dependent on TNFR1 expression and p38 MAPK activity.

Visceral pain is a leading cause of morbidity in gastrointestinal diseases, which is exacerbated by the gut-related side-effects of many analgesics. New treatments are needed and further understanding of the mediators and mechanisms underpinning visceral nociception in disease states is required to facilitate this. The pro-inflammatory cytokine TNFα is linked to pain in both patients with inflammatory bowel disease and irritable bowel syndrome, and has been shown to sensitize colonic sensory neurons. Somatic, TNFα-triggered thermal and mechanical hypersensitivity is mediated by TRPV1 signalling and p38 MAPK activity respectively, downstream of TNFR1 receptor activation. We therefore hypothesized that TNFR1-evoked p38 MAPK activity may also be responsible for TNFα sensitization of colonic afferent responses to the TRPV1 agonist capsaicin, and noxious distension of the bowel. Using Ca2+ imaging of dorsal root ganglion sensory neurons, we observed TNFα-mediated increases in intracellular [Ca2+ ] and sensitization of capsaicin responses. The sensitizing effects of TNFα were dependent on TNFR1 expression and attenuated by p38 MAPK inhibition. Consistent with these findings, ex vivo colonic afferent fibre recordings demonstrated an enhanced response to noxious ramp distention of the bowel and bath application of capsaicin following TNFα pre-treatment. Responses were reversed by p38 MAPK inhibition and absent in tissue from TNFR1 knockout mice. Our findings demonstrate a contribution of TNFR1, p38 MAPK and TRPV1 to TNFα-induced sensitization of colonic afferents, highlighting the potential utility of these drug targets for the treatment of visceral pain in gastrointestinal disease. KEY POINTS: The pro-inflammatory cytokine TNFα is elevated in gastrointestinal disease and sensitizes colonic afferents via modulation of TRPA1 and NaV 1.8 activity. We further develop this understanding by demonstrating a role for p38 MAPK and TRPV1 in TNFα-mediated colonic afferent sensitization. Specifically, we show that: TNFα sensitizes sensory neurons and colonic afferents to the TRPV1 agonist capsaicin. TNFα-mediated sensitization of sensory neurons and colonic nociceptors is dependent on TNFR1 expression. TNFα sensitization of sensory neurons and colonic afferents to capsaicin and noxious ramp distension is abolished by inhibition of p38 MAPK. Collectively these data support the utility of targeting TNFα, TNFR1 and their downstream signalling via p38 MAPK for the treatment of visceral pain in gastrointestinal disease.

Naked Mole-Rats: Resistant to Developing Cancer or Good at Avoiding It?

It is widely accepted that cancer is driven by genetic mutations that confer uncontrolled cell proliferation and tumor formation. For tumors to take hold and grow, cancer cells evolve mechanisms to favorably shape their microenvironment and avoid being cleared by the immune system. Cancer is not unique to human, but rather affects nearly all multicellular organisms albeit to different degrees. The different degrees of cancer susceptibility across the animal kingdom could be attributed to several factors, which have been the subject of several studies in recent years. The naked mole-rat (NMR, Heterocephalus glaber), an exceptionally long-lived rodent, which, as discussed in detail in the next section, displays significant cancer resistance with only a small number of animals being reported to exhibit spontaneous neoplasms. The reason why studying cancer resistance in NMRs is of particular interest is that not only are they now an established laboratory species, but that NMRs are mammals and thus there is great potential for translating knowledge about their cancer resistance into preventing and/or treating cancer in humans and companion animals.

Some Exciting Future Directions for Work on Naked Mole-Rats.

The naked mole-rat is a species of growing research interest. Recent focus on this species from both a biomedical and zoological perspective has led to important discoveries regarding eusociality and ecophysiological and sensory traits associated with life below ground as well as natural protection from variable oxygen availability, acid-induced pain, and the vagaries of aging. These features serve to remind us that many foundational discoveries have arisen using extremophilic organisms and elucidating the mechanisms they employ to survive the harsh environmental conditions they encounter. Investigating these evolved features also facilitates a better understanding of several human disease states that share features with this harsh subterranean milieu. Here, we provide an overview of some unanswered questions and future directions to advance this field, alongside discussion of the tools that could facilitate accelerated progression of research using this enigmatic model.

Sensory Systems of the African Naked Mole-Rat.

Naked mole-rats share some sensory characteristics with other subterraneans, including lack of object vision, retention of the ability to entrain their circadian rhythm to light, and poor hearing. On the other hand, a characteristic that may be specialized in the naked mole-rat is their exquisite orienting responses to the touch of even a single body vibrissa. They have about 100 whisker-like body vibrissae on their otherwise furless bodies. They are also insensitive to chemical and inflammatory pain, likely an adaptation to living in an atmosphere that is high in carbon dioxide, a result of many respiring individuals driving carbon dioxide accumulation. Naked mole-rats have the highest population density among subterranean mammals. High levels of carbon dioxide cause tissue acidosis and associated pain. Remarkably, naked mole-rats are completely immune to carbon dioxide-induced pulmonary edema. However, they retain the ability to detect acid as a taste (sour). Finally, their ability to smell and discriminate odors is comparable to that of rats and mice, but their vomeronasal organ, associated with sensing pheromones, is extremely small and shows a complete lack of post-natal growth. In this chapter, we review what is known about the sensory systems of the naked mole-rat with emphasis on how they differ from other mammals, and even other subterraneans. More extensive accounts of the naked mole-rat's auditory and pain systems can be found in other chapters of this book.

The Somatosensory World of the African Naked Mole-Rat.

The naked mole-rat (Heterocephalus glaber) is famous for its longevity and unusual physiology. This eusocial species that lives in highly ordered and hierarchical colonies with a single breeding queen, also discovered secrets enabling somewhat pain-free living around 20 million years ago. Unlike most mammals, naked mole-rats do not feel the burn of chili pepper's active ingredient, capsaicin, nor the sting of acid. Indeed, by accumulating mutations in genes encoding proteins that are only now being exploited as targets for new pain therapies (the nerve growth factor receptor TrkA and voltage-gated sodium channel, NaV1.7), this species mastered the art of analgesia before humans evolved. Recently, we have identified pain-insensitivity as a trait shared by several closely related African mole-rat species. In this chapter we will show how African mole-rats have evolved pain insensitivity as well as discussing what the proximate factors may have been that led to the evolution of pain-free traits.

African Naked Mole-Rats Demonstrate Extreme Tolerance to Hypoxia and Hypercapnia.

Naked mole-rats are extremely tolerant to low concentrations of oxygen (hypoxia) and high concentrations of carbon dioxide (hypercapnia), which is consistent with the environment that they inhabit. Naked mole-rats combine subterranean living with living in very densely populated colonies where oxygen becomes depleted and carbon dioxide accumulates. In the laboratory, naked mole-rats fully recover from 5 h exposure to 5% O2 and 5 h exposure to 80% CO2, whereas both conditions are rapidly lethal to similarly sized laboratory mice. During anoxia (0% O2) naked mole-rats enter a suspended animation-like state and switch from aerobic metabolism of glucose to anaerobic metabolism of fructose. Additional fascinating characteristics include that naked mole-rats show intrinsic brain tolerance to anoxia; a complete lack of hypoxia-induced and CO2-induced pulmonary edema; and reduced aversion to high concentrations of CO2 and acidic fumes. Here we outline a constellation of physiological and molecular adaptations that correlate with the naked mole-rat's hypoxic/hypercapnic tolerance and which offer potential targets for ameliorating pathological conditions in humans, such as the damage caused during cerebral ischemia.

A stomatin dimer modulates the activity of acid-sensing ion channels.

Stomatin proteins oligomerize at membranes and have been implicated in ion channel regulation and membrane trafficking. To obtain mechanistic insights into their function, we determined three crystal structures of the conserved stomatin domain of mouse stomatin that assembles into a banana-shaped dimer. We show that dimerization is crucial for the repression of acid-sensing ion channel 3 (ASIC3) activity. A hydrophobic pocket at the inside of the concave surface is open in the presence of an internal peptide ligand and closes in the absence of this ligand, and we demonstrate a function of this pocket in the inhibition of ASIC3 activity. In one crystal form, stomatin assembles via two conserved surfaces into a cylindrical oligomer, and these oligomerization surfaces are also essential for the inhibition of ASIC3-mediated currents. The assembly mode of stomatin uncovered in this study might serve as a model to understand oligomerization processes of related membrane-remodelling proteins, such as flotillin and prohibitin.

Atomic force microscopy imaging reveals the formation of ASIC/ENaC cross-clade ion channels.

ASIC and ENaC are co-expressed in various cell types, and there is evidence for a close association between them. Here, we used atomic force microscopy (AFM) to determine whether ASIC1a and ENaC subunits are able to form cross-clade hybrid ion channels. ASIC1a and ENaC could be co-isolated from detergent extracts of tsA 201 cells co-expressing the two subunits. Isolated proteins were incubated with antibodies against ENaC and Fab fragments against ASIC1a. AFM imaging revealed proteins that were decorated by both an antibody and a Fab fragment with an angle of ∼120° between them, indicating the formation of ASIC1a/ENaC heterotrimers.

Skin Aging in Long-Lived Naked Mole-Rats Is Accompanied by Increased Expression of Longevity-Associated and Tumor Suppressor Genes.

Naked mole-rats (NMRs) (Heterocephalus glaber) are long-lived mammals that possess a natural resistance to cancer and other age-related pathologies, maintaining a healthy life span >30 years. In this study, using immunohistochemical and RNA-sequencing analyses, we compare skin morphology, cellular composition, and global transcriptome signatures between young and aged (aged 3‒4 vs. 19‒23 years, respectively) NMRs. We show that similar to aging in human skin, aging in NMRs is accompanied by a decrease in epidermal thickness; keratinocyte proliferation; and a decline in the number of Merkel cells, T cells, antigen-presenting cells, and melanocytes. Similar to that in human skin aging, expression levels of dermal collagens are decreased, whereas matrix metalloproteinase 9 and matrix metalloproteinase 11 levels increased in aged versus in young NMR skin. RNA-sequencing analyses reveal that in contrast to human or mouse skin aging, the transcript levels of several longevity-associated (Igfbp3, Igf2bp3, Ing2) and tumor-suppressor (Btg2, Cdkn1a, Cdkn2c, Dnmt3a, Hic1, Socs3, Sfrp1, Sfrp5, Thbs1, Tsc1, Zfp36) genes are increased in aged NMR skin. Overall, these data suggest that specific features in the NMR skin aging transcriptome might contribute to the resistance of NMRs to spontaneous skin carcinogenesis and provide a platform for further investigations of NMRs as a model organism for studying the biology and disease resistance of human skin.

Selective inflammatory pain insensitivity in the African naked mole-rat (Heterocephalus glaber).

In all mammals, tissue inflammation leads to pain and behavioral sensitization to thermal and mechanical stimuli called hyperalgesia. We studied pain mechanisms in the African naked mole-rat, an unusual rodent species that lacks pain-related neuropeptides (e.g., substance P) in cutaneous sensory fibers. Naked mole-rats show a unique and remarkable lack of pain-related behaviors to two potent algogens, acid and capsaicin. Furthermore, when exposed to inflammatory insults or known mediators, naked mole-rats do not display thermal hyperalgesia. In contrast, naked mole-rats do display nocifensive behaviors in the formalin test and show mechanical hyperalgesia after inflammation. Using electrophysiology, we showed that primary afferent nociceptors in naked mole-rats are insensitive to acid stimuli, consistent with the animal's lack of acid-induced behavior. Acid transduction by sensory neurons is observed in birds, amphibians, and fish, which suggests that this tranduction mechanism has been selectively disabled in the naked mole-rat in the course of its evolution. In contrast, nociceptors do respond vigorously to capsaicin, and we also show that sensory neurons express a transient receptor potential vanilloid channel-1 ion channel that is capsaicin sensitive. Nevertheless, the activation of capsaicin-sensitive sensory neurons in naked mole-rats does not produce pain-related behavior. We show that capsaicin-sensitive nociceptors in the naked mole-rat are functionally connected to superficial dorsal horn neurons as in mice. However, the same nociceptors are also functionally connected to deep dorsal horn neurons, a connectivity that is rare in mice. The pain biology of the naked mole-rat is unique among mammals, thus the study of pain mechanisms in this unusual species can provide major insights into what constitutes "normal" mammalian nociception.

Modulation of acid-sensing ion channel activity by nitric oxide.

Acid-sensing ion channels (ASICs) are a class of ion channels activated by extracellular protons and are believed to mediate the pain caused by tissue acidosis. Although ASICs have been widely studied, little is known about their regulation by inflammatory mediators. Here, we provide evidence that nitric oxide (NO) potentiates the activity of ASICs. Whole-cell patch-clamp recordings were performed on neonatal rat cultured dorsal root ganglion neurons and on ASIC isoforms expressed in CHO cells. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) potentiates proton-gated currents in DRG neurons and proton-gated currents in CHO cells expressing each of the acid-sensitive ASIC subunits. Modulators of the cGMP/PKG pathway had no effect on the potentiation, but in excised patches from CHO cells expressing ASIC2a, the potentiation could be reversed by externally applied reducing agents. NO therefore has a direct external effect on the ASIC ion channel, probably through oxidization of cysteine residues. Complementary psychophysiological studies were performed using iontophoresis of acidic solutions through the skin of human volunteers. Topical application of the NO donor glyceryl trinitrate significantly increased acid-evoked pain but did not affect heat or mechanical pain thresholds. ASICs may therefore play an important role in the pain associated with metabolic stress and inflammation, where both tissue acidosis and a high level of NO are present.

Proton binding sites involved in the activation of acid-sensing ion channel ASIC2a.

Most acid-sensing ion channel (ASIC) subunits are activated by protons, but ASIC2b (a splice variant of ASIC2a) is acid-insensitive. Differences in protonatable residues between the extracellular loop regions of ASIC2a and ASIC2b may explain this difference. Site-directed mutagenesis, combined with immunocytochemistry and whole-cell patch clamp, demonstrated that mutating any one of five ASIC2a sites produces channels that traffic normally to the cell surface membrane but are insensitive to protons. One of the mutants forms functional heteromers with ASIC1a and ASIC2a, demonstrating that ion transport is intact in this mutant. These five sites may be involved in the activation of ASIC2a by protons.

Fructose-driven glycolysis supports anoxia resistance in the naked mole-rat.

The African naked mole-rat's (Heterocephalus glaber) social and subterranean lifestyle generates a hypoxic niche. Under experimental conditions, naked mole-rats tolerate hours of extreme hypoxia and survive 18 minutes of total oxygen deprivation (anoxia) without apparent injury. During anoxia, the naked mole-rat switches to anaerobic metabolism fueled by fructose, which is actively accumulated and metabolized to lactate in the brain. Global expression of the GLUT5 fructose transporter and high levels of ketohexokinase were identified as molecular signatures of fructose metabolism. Fructose-driven glycolytic respiration in naked mole-rat tissues avoids feedback inhibition of glycolysis via phosphofructokinase, supporting viability. The metabolic rewiring of glycolysis can circumvent the normally lethal effects of oxygen deprivation, a mechanism that could be harnessed to minimize hypoxic damage in human disease.

Ear Structures of the Naked Mole-Rat, Heterocephalus glaber, and Its Relatives (Rodentia: Bathyergidae).

Although increasingly popular as a laboratory species, very little is known about the peripheral auditory system of the naked mole-rat, Heterocephalus glaber. In this study, middle and inner ears of naked mole-rats of a range of ages were examined using micro-computed tomography and dissection. The ears of five other bathyergid species (Bathyergus suillus, Cryptomys hottentotus, Fukomys micklemi, Georychus capensis and Heliophobius argenteocinereus) were examined for comparative purposes. The middle ears of bathyergids show features commonly found in other members of the Ctenohystrica rodent clade, including a fused malleus and incus, a synovial stapedio-vestibular articulation and the loss of the stapedius muscle. Heterocephalus deviates morphologically from the other bathyergids examined in that it has a more complex mastoid cavity structure, poorly-ossified processes of the malleus and incus, a 'columelliform' stapes and fewer cochlear turns. Bathyergids have semicircular canals with unusually wide diameters relative to their radii of curvature. How the lateral semicircular canal reaches the vestibule differs between species. Heterocephalus has much more limited high-frequency hearing than would be predicted from its small ear structures. The spongy bone forming its ossicular processes, the weak incudo-stapedial articulation, the columelliform stapes and (compared to other bathyergids) reduced cochlear coiling are all potentially degenerate features which might reflect a lack of selective pressure on its peripheral auditory system. Substantial intraspecific differences were found in certain middle and inner ear structures, which might also result from relaxed selective pressures. However, such interpretations must be treated with caution in the absence of experimental evidence.

Hypofunctional TrkA Accounts for the Absence of Pain Sensitization in the African Naked Mole-Rat.

The naked mole-rat is a subterranean rodent lacking several pain behaviors found in humans, rats, and mice. For example, nerve growth factor (NGF), an important mediator of pain sensitization, fails to produce thermal hyperalgesia in naked mole-rats. The sensitization of capsaicin-sensitive TRPV1 ion channels is necessary for NGF-induced hyperalgesia, but naked mole-rats have fully functional TRPV1 channels. We show that exposing isolated naked mole-rat nociceptors to NGF does not sensitize TRPV1. However, the naked mole-rat NGF receptor TrkA displays a reduced ability to engage signal transduction pathways that sensitize TRPV1. Between one- and three-amino-acid substitutions in the kinase domain of the naked mole-rat TrkA are sufficient to render the receptor hypofunctional, and this is associated with the absence of heat hyperalgesia. Our data suggest that evolution has selected for a TrkA variant that abolishes a robust nociceptive behavior in this species but is still compatible with species fitness.