Susanne Holtze

Analysis of Alpha-2 Macroglobulin from the Long-Lived and Cancer-Resistant Naked Mole-Rat and Human Plasma

Background The naked mole-rat (NMR) is a long-lived and cancer resistant species. Identification of potential anti-cancer and age related mechanisms is of great interest and makes this species eminent to investigate anti-cancer strategies and understand aging mechanisms. Since it is known that the NMR expresses higher liver mRNA-levels of alpha 2-macroglobulin than mice, nothing is known about its structure, functionality or expression level in the NMR compared to the human A2M. Results Here we show a comprehensive analysis of NMR- and human plasma-A2M, showing a different prediction in glycosylation of NMR-A2M, which results in a higher molecular weight compared to human A2M. Additionally, we found a higher concentration of A2M (8.3±0.44 mg/mL vs. and 4.4±0.20 mg/mL) and a lower total plasma protein content (38.7±1.79 mg/mL vs. 61.7±3.20 mg/mL) in NMR compared to human. NMR-A2M can be transformed by methylamine and trypsin resulting in a conformational change similar to human A2M. NMR-A2M is detectable by a polyclonal antibody against human A2M. Determination of tryptic and anti-tryptic activity of NMR and human plasma revealed a higher anti-tryptic activity of the NMR plasma. On the other hand, less proteolytic activity was found in NMR plasma compared to human plasma. Conclusion We found transformed NMR-A2M binding to its specific receptor LRP1. We could demonstrate lower protein expression of LRP1 in the NMR liver tissue compared to human but higher expression of A2M. This was accompanied by a higher EpCAM protein expression as central adhesion molecule in cancer progression. NMR-plasma was capable to increase the adhesion in human fibroblast in vitro most probably by increasing CD29 protein expression. This is the first report, demonstrating similarities as well as distinct differences between A2M in NMR and human plasma. This might be directly linked to the intriguing phenotype of the NMR and suggests that A2M might probably play an important role in anti-cancer and the anti-aging mechanisms in the NMR.

Low sulfide levels and a high degree of cystathionine β-synthase (CBS) activation by S-adenosylmethionine (SAM) in the long-lived naked mole-rat.

Hydrogen sulfide (H2S) is a gaseous signalling molecule involved in many physiological and pathological processes. There is increasing evidence that H2S is implicated in aging and lifespan control in the diet-induced longevity models. However, blood sulfide concentration of naturally long-lived species is not known. Here we measured blood sulfide in the long-lived naked mole-rat and five other mammalian species considerably differing in lifespan and found a negative correlation between blood sulfide and maximum longevity residual. In addition, we show that the naked mole-rat cystathionine β-synthase (CBS), an enzyme whose activity in the liver significantly contributes to systemic sulfide levels, has lower activity in the liver and is activated to a higher degree by S-adenosylmethionine compared to other species. These results add complexity to the understanding of the role of H2S in aging and call for detailed research on naked mole-rat transsulfuration.

Unraveling the gut microbiome of the long-lived naked mole-rat

The naked mole-rat (Heterocephalus glaber) is a subterranean mouse-sized African mammal that shows astonishingly few age-related degenerative changes and seems to not be affected by cancer. These features make this wild rodent an excellent model to study the biology of healthy aging and longevity. Here we characterize for the first time the intestinal microbial ecosystem of the naked mole-rat in comparison to humans and other mammals, highlighting peculiarities related to the specific living environment, such as the enrichment in bacteria able to utilize soil sulfate as a terminal electron acceptor to sustain an anaerobic oxidative metabolism. Interestingly, some compositional gut microbiota peculiarities were also shared with human gut microbial ecosystems of centenarians and Hadza hunter-gatherers, considered as models of a healthy gut microbiome and of a homeostatic and highly adaptive gut microbiota-host relationship, respectively. In addition, we found an enrichment of short-chain fatty acids and carbohydrate degradation products in naked mole-rat compared to human samples. These data confirm the importance of the gut microbial ecosystem as an adaptive partner for the mammalian biology and health, independently of the host phylogeny.

Study of age-dependent structural and functional changes of mitochondria in skeletal muscles and heart of naked mole rats (Heterocephalus glaber)

Morphometric analysis of mitochondria in skeletal muscles and heart of 6- and 60-month-old naked mole rats (Heterocephalus glaber) revealed a significant age-dependent increase in the total area of mitochondrial cross-sections in studied muscle fibers. For 6- and 60-month-old animals, these values were 4.8 ± 0.4 and 12.7 ± 1.8%, respectively. This effect is mainly based on an increase in the number of mitochondria. In 6-month-old naked mole rats, there were 0.23 ± 0.02 mitochondrial cross-sections per μm2 of muscle fiber, while in 60-month-old animals this value was 0.47 ± 0.03. The average area of a single mitochondrial cross-section also increased with age in skeletal muscles–from 0.21 ± 0.01 to 0.29 ± 0.03 μm2. Thus, naked mole rats show a drastic enlargement of the mitochondrial apparatus in skeletal muscles with age due to an increase in the number of mitochondria and their size. They possess a neotenic type of chondriome accompanied by specific features of mitochondrial functioning in the state of oxidative phosphorylation and a significant decrease in the level of matrix adenine nucleotides.

Monitoring health and reproductive status of olms (Proteus anguinus)by ultrasound

The olm (Proteus anguinus) is a troglomorphic, neotenous amphibian with extraordinary life expectancy and unique adaptations that deserve further investigation. A low reproductive rate and habitat decline render it threatened by extinction. Establishing captive populations for maintenance and artificial breeding may one day become crucial to the species. Longitudinal, in-vivo assessment of inner organs is invaluable to our understanding of reproductive physiology, health, and behavior. Using ultrasound, we measured heart rate and assessed health and reproductive status of 13 captive olms at Zagreb Zoo. Heart rate averaged 42.9 ± 4.6 bpm (32–55 bpm), as determined via pulsed-wave Doppler at 4–12 MHz. By using frequencies of up to 70 MHz (ultrasound biomicroscopy), inner organs were visualized in detail. Assessment of the gastrointestinal tract provided insights into feeding status and digestive processes. Several subclinical pathologies were detected, including biliary sludge, subcutaneous edema, ascites, and skin lesions. Detection of skin lesions by ultrasound was more sensitive than visual adspection. Olms with ultrasonographically detected skin lesions tested positive for Saprolegnia and were treated. Three of the four affected individuals survived and subsequently tested negative for Saprolegnia. Sex was reliably determined; only one individual proved male. The reason for this extreme female-biased sex-ratio remains unknown. However, as most of the individuals were flushed from the caves by strong currents in spring, the sample may not be representative of natural populations. In female olms, different stages of ovarian follicular development were observed with diameters ranging between 0.1 and 1.1 mm. Results were confirmed by comparing ultrasound, necropsy, and histological findings of one dead specimen. In summary, ultrasound proved a valuable tool to support conservation and captive breeding programs by allowing non-invasive assessment of physiological parameters, clinical condition, and reproductive status in olms.

Embryos and embryonic stem cells from the white rhinoceros

The northern white rhinoceros (NWR, Ceratotherium simum cottoni) is the most endangered mammal in the world with only two females surviving. Here we adapt existing assisted reproduction techniques (ART) to fertilize Southern White Rhinoceros (SWR) oocytes with NWR spermatozoa. We show that rhinoceros oocytes can be repeatedly recovered from live SWR females by transrectal ovum pick-up, matured, fertilized by intracytoplasmic sperm injection and developed to the blastocyst stage in vitro. Next, we generate hybrid rhinoceros embryos in vitro using gametes of NWR and SWR. We also establish embryonic stem cell lines from the SWR blastocysts. Blastocysts are cryopreserved for later embryo transfer. Our results indicate that ART could be a viable strategy to rescue genes from the iconic, almost extinct, northern white rhinoceros and may also have broader impact if applied with similar success to other endangered large mammalian species.The Southern (SWR) and Northern (NWR) are two subspecies of the White Rhinoceros with the NWR being almost extinct. Here, using assisted reproduction technology, the authors produce and cryopreserve SWR purebred and NWR-SWR hybrid embryos developed to the blastocyst stage, and also generate embryonic stem cell lines, in an attempt to save genes of the NWR.

Long-lived rodents reveal signatures of positive selection in genes associated with lifespan

The genetics of lifespan determination is poorly understood. Most research has been done on short-lived animals and it is unclear if these insights can be transferred to long-lived mammals like humans. Some African mole-rats (Bathyergidae) have life expectancies that are multiple times higher than similar sized and phylogenetically closely related rodents. To gain new insights into genetic mechanisms determining mammalian lifespans, we obtained genomic and transcriptomic data from 17 rodent species and scanned eleven evolutionary branches associated with the evolution of enhanced longevity for positively selected genes (PSGs). Indicating relevance for aging, the set of 250 identified PSGs showed in liver of long-lived naked mole-rats and short-lived rats an expression pattern that fits the antagonistic pleiotropy theory of aging. Moreover, we found the PSGs to be enriched for genes known to be related to aging. Among these enrichments were “cellular respiration” and “metal ion homeostasis”, as well as functional terms associated with processes regulated by the mTOR pathway: translation, autophagy and inflammation. Remarkably, among PSGs are RHEB, a regulator of mTOR, and IGF1, both central components of aging-relevant pathways, as well as genes yet unknown to be aging-associated but representing convincing functional candidates, e.g. RHEBL1, AMHR2, PSMG1 and AGER. Exemplary protein homology modeling suggests functional consequences for amino acid changes under positive selection. Therefore, we conclude that our results provide a meaningful resource for follow-up studies to mechanistically link identified genes and amino acids under positive selection to aging and lifespan determination.

Naked mole-rat transcriptome signatures of socially suppressed sexual maturation and links of reproduction to aging

BackgroundNaked mole-rats (NMRs) are eusocially organized in colonies. Although breeders carry the additional metabolic load of reproduction, they are extremely long-lived and remain fertile throughout their lifespan. This phenomenon contrasts the disposable soma theory of aging stating that organisms can invest their resources either in somatic maintenance, enabling a longer lifespan, or in reproduction, at the cost of longevity. Here, we present a comparative transcriptome analysis of breeders vs. non-breeders of the eusocial, long-lived NMR vs. the polygynous and shorter-lived guinea pig (GP).ResultsComparative transcriptome analysis of tissue samples from ten organs showed, in contrast to GPs, low levels of differentiation between sexes in adult NMR non-breeders. After transition into breeders, NMR transcriptomes are markedly sex-specific, show pronounced feedback signaling via gonadal steroids, and have similarities to reproductive phenotypes in African cichlid fish, which also exhibit social status changes between dominant and subordinate phenotypes. Further, NMRs show functional enrichment of status-related expression differences associated with aging. Lipid metabolism and oxidative phosphorylation—molecular networks known to be linked to aging—were identified among most affected gene sets. Remarkably and in contrast to GPs, transcriptome patterns associated with longevity are reinforced in NMR breeders.ConclusionOur results provide comprehensive and unbiased molecular insights into interspecies differences between NMRs and GPs, both in sexual maturation and in the impact of reproduction on longevity. We present molecular evidence that sexual maturation in NMRs is socially suppressed. In agreement with evolutionary theories of aging in eusocial organisms, we have identified transcriptome patterns in NMR breeders that—in contrast to the disposable soma theory of aging—may slow down aging rates and potentially contribute to their exceptional long life- and healthspan.

Species comparison of liver proteomes reveals links to naked mole-rat longevity and human aging

BackgroundMammals display a wide range of variation in their lifespan. Investigating the molecular networks that distinguish long- from short-lived species has proven useful to identify determinants of longevity. Here, we compared the livers of young and old long-lived naked mole-rats (NMRs) and the phylogenetically closely related, shorter-lived, guinea pigs using an integrated omics approach.ResultsWe found that NMR livers display a unique expression pattern of mitochondrial proteins that results in distinct metabolic features of their mitochondria. For instance, we observed a generally reduced respiration rate associated with lower protein levels of respiratory chain components, particularly complex I, and increased capacity to utilize fatty acids. Interestingly, we show that the same molecular networks are affected during aging in both NMRs and humans, supporting a direct link to the extraordinary longevity of both species. Finally, we identified a novel detoxification pathway linked to longevity and validated it experimentally in the nematode Caenorhabditis elegans.ConclusionsOur work demonstrates the benefits of integrating proteomic and transcriptomic data to perform cross-species comparisons of longevity-associated networks. Using a multispecies approach, we show at the molecular level that livers of NMRs display progressive age-dependent changes that recapitulate typical signatures of aging despite the negligible senescence and extraordinary longevity of these rodents.

Non-Breeding Eusocial Mole-Rats Produce Viable Sperm—Spermiogram and Functional Testicular Morphology of Fukomys anselli

Ansell’s mole-rats (Fukomys anselli) are subterranean rodents living in families composed of about 20 members with a single breeding pair and their non-breeding offspring. Most of them remain with their parents for their lifetime and help to maintain and defend the natal burrow system, forage, and care for younger siblings. Since incest avoidance is based on individual recognition (and not on social suppression) we expect that non-breeders produce viable sperm spontaneously. We compared the sperm of breeding and non-breeding males, obtained by electroejaculation and found no significant differences in sperm parameters between both groups. Here, we used electroejaculation to obtain semen for the first time in a subterranean mammal. Spermiogram analysis revealed no significant differences in sperm parameters between breeders and non-breeders. We found significantly larger testes (measured on autopsies and on living animals per ultrasonography) of breeders compared to non-breeders (with body mass having a significant effect). There were no marked histological differences between breeding and non-breeding males, and the relative area occupied by Leydig cells and seminiferous tubules on histological sections, respectively, was not significantly different between both groups. The seminiferous epithelium and to a lesser degree the interstitial testicular tissue are characterized by lesions (vacuolar degenerations), however, this feature does not hinder fertilization even in advanced stages of life. The continuous production of viable sperm also in sexually abstinent non-breeders might be best understood in light of the mating and social system of Fukomys anselli, and the potential to found a new family following an unpredictable and rare encounter with an unfamiliar female (“provoked or induced dispersal”). Apparently, the non-breeders do not reproduce because they do not copulate but not because they would be physiologically infertile. The significantly increased testes volume of breeding males (compared to non-breeders) is in agreement with previously found higher testosterone levels of breeders.