Insights into Sex Chromosome Evolution and Aging from the Genome of a Short-Lived Fish.

The killifish Nothobranchius furzeri is the shortest-lived vertebrate that can be bred in the laboratory. Its rapid growth, early sexual maturation, fast aging, and arrested embryonic development (diapause) make it an attractive model organism in biomedical research. Here, we report a draft sequence of its genome that allowed us to uncover an intra-species Y chromosome polymorphism representing-in real time-different stages of sex chromosome formation that display features of early mammalian XY evolution "in action." Our data suggest that gdf6Y, encoding a TGF-ß family growth factor, is the master sex-determining gene in N. furzeri. Moreover, we observed genomic clustering of aging-related genes, identified genes under positive selection, and revealed significant similarities of gene expression profiles between diapause and aging, particularly for genes controlling cell cycle and translation. The annotated genome sequence is provided as an online resource (http://www.nothobranchius.info/NFINgb).

RNAseq Analysis of Brain Aging in Wild Specimens of Short-Lived Turquoise Killifish: Commonalities and Differences With Aging Under Laboratory Conditions.

A vast body of studies is available that describe age-dependent gene expression in relation to aging in a number of different model species. These data were obtained from animals kept in conditions with reduced environmental challenges, abundant food, and deprivation of natural sensory stimulation. Here, we compared wild- and captive aging in the short-lived turquoise killifish (Nothobranchius furzeri). These fish inhabit temporary ponds in the African savannah. When the ponds are flooded, eggs hatch synchronously, enabling a precise timing of their individual and population age. We collected the brains of wild fish of different ages and quantified the global age-dependent regulation of transcripts using RNAseq. A major difference between captive and wild populations is that wild populations had unlimited access to food and hence grew to larger sizes and reached asymptotic size more rapidly, enabling the analysis of age-dependent gene expression without the confounding effect of adult brain growth. We found that the majority of differentially expressed genes show the same direction of regulation in wild and captive populations. However, a number of genes were regulated in opposite direction. Genes downregulated in the wild and upregulated in captivity were enriched for terms related to neuronal communication. Genes upregulated in the wild and downregulated in captive conditions were enriched in terms related to DNA replication. Finally, the rate of age-dependent gene regulation was higher in wild animals, suggesting a phenomenon of accelerated aging.

Localization and Characterization of Major Neurogenic Niches in the Brain of the Lesser-Spotted Dogfish Scyliorhinus canicula.

Adult neurogenesis is defined as the ability of specialized cells in the postnatal brain to produce new functional neurons and to integrate them into the already-established neuronal network. This phenomenon is common in all vertebrates and has been found to be extremely relevant for numerous processes, such as long-term memory, learning, and anxiety responses, and it has been also found to be involved in neurodegenerative and psychiatric disorders. Adult neurogenesis has been studied extensively in many vertebrate models, from fish to human, and observed also in the more basal cartilaginous fish, such as the lesser-spotted dogfish, Scyliorhinus canicula, but a detailed description of neurogenic niches in this animal is, to date, limited to the telencephalic areas. With this article, we aim to extend the characterization of the neurogenic niches of S. canicula in other main areas of the brain: we analyzed via double immunofluorescence sections of telencephalon, optic tectum, and cerebellum with markers of proliferation (PCNA) and mitosis (pH3) in conjunction with glial cell (S100ß) and stem cell (Msi1) markers, to identify the actively proliferating cells inside the neurogenic niches. We also labeled adult postmitotic neurons (NeuN) to exclude double labeling with actively proliferating cells (PCNA). Lastly, we observed the presence of the autofluorescent aging marker, lipofuscin, contained inside lysosomes in neurogenic areas.

Distribution of Brain-Derived Neurotrophic Factor in the Brain of the Small-Spotted Catshark Scyliorhinus canicula, and Evolution of Neurotrophins in Basal Vertebrates.

Neurotrophins (NTFs) are structurally related neurotrophic factors essential for differentiation, survival, neurite outgrowth, and the plasticity of neurons. Abnormalities associated with neurotrophin-signaling (NTF-signaling) were associated with neuropathies, neurodegenerative disorders, and age-associated cognitive decline. Among the neurotrophins, brain-derived neurotrophic factor (BDNF) has the highest expression and is expressed in mammals by specific cells throughout the brain, with particularly high expression in the hippocampus and cerebral cortex. Whole genome sequencing efforts showed that NTF signaling evolved before the evolution of Vertebrates; thus, the shared ancestor of Protostomes, Cyclostomes, and Deuterostomes must have possessed a single ortholog of neurotrophins. After the first round of whole genome duplication that occurred in the last common ancestor of Vertebrates, the presence of two neurotrophins in Agnatha was hypothesized, while the monophyletic group of cartilaginous fishes, or Chondrichthyans, was situated immediately after the second whole genome duplication round that occurred in the last common ancestor of Gnathostomes. Chondrichthyans represent the outgroup of all other living jawed vertebrates (Gnathostomes) and the sister group of Osteichthyans (comprehensive of Actinopterygians and Sarcopterygians). We were able to first identify the second neurotrophin in Agnatha. Secondly, we expanded our analysis to include the Chondrichthyans, with their strategic phylogenetic position as the most basal extant Gnathostome taxon. Results from the phylogenetic analysis confirmed the presence of four neurotrophins in the Chondrichthyans, namely the orthologs of the four mammalian neurotrophins BDNF, NGF, NT-3, and NT-4. We then proceeded to study the expression of BDNF in the adult brain of the Chondrichthyan Scyliorhinus canicula. Our results showed that BDNF is highly expressed in the S. canicula brain and that its expression is highest in the Telencephalon, while the Mesencephalic and Diencephalic areas showed expression of BDNF in isolated and well-defined cell groups. NGF was expressed at much lower levels that could be detected by PCR but not by in situ hybridization. Our results warrant further investigations in Chondrichthyans to characterize the putative ancestral function of neurotrophins in Vertebrates.

MiR-29 coordinates age-dependent plasticity brakes in the adult visual cortex.

Visual cortical circuits show profound plasticity during early life and are later stabilized by molecular "brakes" limiting excessive rewiring beyond a critical period. The mechanisms coordinating the expression of these factors during the transition from development to adulthood remain unknown. We found that miR-29a expression in the visual cortex dramatically increases with age, but it is not experience-dependent. Precocious high levels of miR-29a blocked ocular dominance plasticity and caused an early appearance of perineuronal nets. Conversely, inhibition of miR-29a in adult mice using LNA antagomirs activated ocular dominance plasticity, reduced perineuronal nets, and restored their juvenile chemical composition. Activated adult plasticity had the typical functional and proteomic signature of critical period plasticity. Transcriptomic and proteomic studies indicated that miR-29a manipulation regulates the expression of plasticity brakes in specific cortical circuits. These data indicate that miR-29a is a regulator of the plasticity brakes promoting age-dependent stabilization of visual cortical connections.

The sources of sex differences in aging in annual fishes.

Intersexual differences in life span (age at death) and aging (increase in mortality risk associated with functional deterioration) are widespread among animals, from nematodes to humans. Males often live shorter than females, but there is substantial unexplained variation among species and populations. Despite extensive research, it is poorly understood how life span differences between the sexes are modulated by an interplay among genetic, environmental and social factors. The goal of our study was to test how sex differences in life span and ageing are modulated by social and environmental factors, and by intrinsic differences between males and females. To disentangle the complex basis of sex differences in life span and aging, we combined comparative data from sex ratios in 367 natural populations of four species of African annual killifish with experimental results on sex differences in life span and aging from eight laboratory populations tested in treatments that varied social and environmental conditions. In the wild, females consistently outlived males. In captivity, sex-specific mortality depended on social conditions. In social-housed experimental groups, male-biased mortality persisted in two aggressive species, but ceased in two placid species. When social and physical contacts were prevented by housing all fish individually, male-biased mortality ceased in all four species. This outcome held across benign and challenging environmental conditions. Fitting demographic survival models revealed that increased baseline mortality was primarily responsible for a shorter male life span in social-housing conditions. The timing and rate of aging were not different between the sexes. No marker of functional aging we recorded in our study (lipofuscin accumulation, proliferative changes in kidney and liver) differed between males and females, despite their previously confirmed association with functional aging in Nothobranchius killifish. We show that sex differences in life span and aging in killifish are driven by a combination of social and environmental conditions, rather than differential functional aging. They are primarily linked to sexual selection but precipitated through multiple processes (predation, social interference). This demonstrates how sex-specific mortality varies among species even within an ecologically and evolutionary discrete lineage and explains how external factors mediate this difference.

Membrane lipids and maximum lifespan in clownfish.

The longevity-homeoviscous adaptation (LHA) theory of ageing states that lipid composition of cell membranes is linked to metabolic rate and lifespan, which has been widely shown in mammals and birds but not sufficiently in fish. In this study, two species of the genus Amphiprion (Amphiprion percula and Amphiprion clarkii, with estimated maximum lifespan potentials [MLSP] of 30 and 9-16 years, respectively) and the damselfish Chromis viridis (estimated MLSP of 1-2 years) were chosen to test the LHA theory of ageing in a potential model of exceptional longevity. Brain, livers and samples of skeletal muscle were collected for lipid analyses and integral part in the computation of membrane peroxidation indexes (PIn) from phospholipid (PL) fractions and PL fatty acid composition. When only the two Amphiprion species were compared, results pointed to the existence of a negative correlation between membrane PIn value and maximum lifespan, well in line with the predictions from the LHA theory of ageing. Nevertheless, contradictory data were obtained when the two Amphiprion species were compared to the shorter-lived C. viridis. These results along with those obtained in previous studies on fish denote that the magnitude (and sometimes the direction) of the differences observed in membrane lipid composition and peroxidation index with MLSP cannot explain alone the diversity in longevity found among fishes.

Reduced proteasome activity in the aging brain results in ribosome stoichiometry loss and aggregation.

A progressive loss of protein homeostasis is characteristic of aging and a driver of neurodegeneration. To investigate this process quantitatively, we characterized proteome dynamics during brain aging in the short-lived vertebrate Nothobranchius furzeri combining transcriptomics and proteomics. We detected a progressive reduction in the correlation between protein and mRNA, mainly due to post-transcriptional mechanisms that account for over 40% of the age-regulated proteins. These changes cause a progressive loss of stoichiometry in several protein complexes, including ribosomes, which show impaired assembly/disassembly and are enriched in protein aggregates in old brains. Mechanistically, we show that reduction of proteasome activity is an early event during brain aging and is sufficient to induce proteomic signatures of aging and loss of stoichiometry in vivo. Using longitudinal transcriptomic data, we show that the magnitude of early life decline in proteasome levels is a major risk factor for mortality. Our work defines causative events in the aging process that can be targeted to prevent loss of protein homeostasis and delay the onset of age-related neurodegeneration.

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.

What have we learned on aging from omics studies?

Aging is a complex process. Transcriptomic studies of the last decade have identified genes and pathways that are regulated during aging in multiple species and organs. Yet, since a manifold of pathways are regulated and the amplitude of regulation is often small, reproducibility across studies is moderate and disentangling cause-consequence relationships has proven challenging. Here, we review a number of consistent findings in the light of more recent, longitudinal studies and of studies combining transcriptomics and proteomics that identified deregulation of protein biosynthetic pathways as an early event and likely driver of aging.

The companion dog as a unique translational model for aging.

The dog is a unique species due to its wide variation among breeds in terms of size, morphology, behaviour and lifespan, coupled with a genetic structure that facilitates the dissection of the genetic architecture that controls these traits. Dogs and humans co-evolved and share recent evolutionary selection processes, such as adaptation to digest starch-rich diets. Many diseases of the dog have a human counterpart, and notably Alzheimer's disease, which is otherwise difficult to model in other organisms. Unlike laboratory animals, companion dogs share the human environment and lifestyle, are exposed to the same pollutants, and are faced with pathogens and infections. Dogs represented a very useful model to understand the relationship between size, insulin-like growth factor-1 genetic variation and lifespan, and have been used to test the effects of dietary restriction and immunotherapy for Alzheimer's disease. Very recently, rapamycin was tested in companion dogs outside the laboratory, and this approach where citizens are involved in research aimed at the benefit of dog welfare might become a game changer in geroscience.

Analysis of the coding sequences of clownfish reveals molecular convergence in the evolution of lifespan.

BACKGROUND: Standard evolutionary theories of aging postulate that reduced extrinsic mortality leads to evolution of longevity. Clownfishes of the genus Amphiprion live in a symbiotic relationship with sea anemones that provide protection from predators. We performed a survey and identified at least two species with a lifespan of over 20 years. Given their small size and ease of captive reproduction, clownfish lend themselves as experimental models of exceptional longevity. To identify genetic correlates of exceptional longevity, we sequenced the transcriptomes of Amphiprion percula and A. clarkii and performed a scan for positively-selected genes (PSGs). RESULTS: The PSGs that we identified in the last common clownfish ancestor were compared with PSGs detected in long-lived mole rats and short-lived killifishes revealing convergent evolution in processes such as mitochondrial biogenesis. Among individual genes, the Mitochondrial Transcription Termination Factor 1 (MTERF1), was positively-selected in all three clades, whereas the Glutathione S-Transferase Kappa 1 (GSTK1) was under positive selection in two independent clades. For the latter, homology modelling strongly suggested that positive selection targeted enzymatically important residues. CONCLUSIONS: These results indicate that specific pathways were recruited in independent lineages evolving an exceptionally extended or shortened lifespan and point to mito-nuclear balance as a key factor.

Correction to: A miRNA catalogue and ncRNA annotation of the short-living fish Nothobranchius furzeri.

Following the publication of this article [1], the authors reported that the images of Figs. 1, 2 and 3 were published in the incorrect order, whereby they mismatch with their captions.

Outgroups and Positive Selection: The Nothobranchius furzeri Case.

Applications of positive selection analysis increase with the number of species for which genome/transcriptome sequences become available. Using the recently sequenced turquoise killifish (Nothobranchius furzeri) genome as an example, we compare two different approaches based on different outgroup selection. The combination of these two methods allows the origin of positively selected sites in aging-related genes of the N. furzeri genome to be determined.

Limited scope for reproductive senescence in wild populations of a short-lived fish.

Senescence in wild populations was long considered negligible but current evidence suggests that it is widespread in natural populations of mammals and birds, affecting the survival and reproductive output of older individuals. In contrast, little is known about reproductive senescence in species with asymptotic growth that can keep increasing their reproductive output as they grow older and larger. Using a cross-sectional study, we tested age-related decline in fecundity and relative allocation to reproduction in five wild populations of an annual killifish, Nothobranchius furzeri (Cyprinodontiformes). We did not detect any decline in absolute female egg production over their short lifespan in the wild. Relative fecundity (egg production controlled for female body mass) tended to decrease with age. This effect was driven primarily by a single population that survived 17 weeks, almost twice as long as the median persistence of the other four study populations. There was no decrease in relative ovary mass while in males, relative testes mass actually increased with age. Intra-population variation in relative ovary mass increased in older females suggesting heterogeneity in individual trajectories of female reproductive allocation. Overall, we demonstrate that annual killifish do not experience significant age-related decline in reproductive functions during their very short lifespan in the wild despite the marked deterioration of gonad tissue detected in captivity.

MicroRNA miR-29 controls a compensatory response to limit neuronal iron accumulation during adult life and aging.

BACKGROUND: A widespread modulation of gene expression occurs in the aging brain, but little is known as to the upstream drivers of these changes. MicroRNAs emerged as fine regulators of gene expression in many biological contexts and they are modulated by age. MicroRNAs may therefore be part of the upstream drivers of the global gene expression modulation correlated with aging and aging-related phenotypes. RESULTS: Here, we show that microRNA-29 (miR-29) is induced during aging in short-lived turquoise killifish brain and genetic antagonism of its function induces a gene-expression signature typical of aging. Mechanicistically, we identified Ireb2 (a master gene for intracellular iron delivery that encodes for IRP2 protein), as a novel miR-29 target. MiR-29 is induced by iron loading and, in turn, it reduces IRP2 expression in vivo, therefore limiting intracellular iron delivery in neurons. Genetically modified fish with neuro-specific miR-29 deficiency exhibit increased levels of IRP2 and transferrin receptor, increased iron content, and oxidative stress. CONCLUSIONS: Our results demonstrate that age-dependent miR-29 upregulation is an adaptive mechanism that counteracts the expression of some aging-related phenotypes and its anti-aging activity is primarily exerted by regulating intracellular iron homeostasis limiting excessive iron-exposure in neurons.

A miRNA catalogue and ncRNA annotation of the short-living fish Nothobranchius furzeri.

BACKGROUND: The short-lived fish Nothobranchius furzeri is the shortest-lived vertebrate that can be cultured in captivity and was recently established as a model organism for aging research. Small non-coding RNAs, especially miRNAs, are implicated in age dependent control of gene expression. RESULTS: Here, we present a comprehensive catalogue of miRNAs and several other non-coding RNA classes (ncRNAs) for Nothobranchius furzeri. Analyzing multiple small RNA-Seq libraries, we show most of these identified miRNAs are expressed in at least one of seven Nothobranchius species. Additionally, duplication and clustering of N. furzeri miRNAs was analyzed and compared to the four fish species Danio rerio, Oryzias latipes, Gasterosteus aculeatus and Takifugu rubripes. A peculiar characteristic of N. furzeri, as compared to other teleosts, was a duplication of the miR-29 cluster. CONCLUSION: The completeness of the catalogue we provide is comparable to that of the zebrafish. This catalogue represents a basis to investigate the role of miRNAs in aging and development in this species.

Pregnant Women's Preferences for Men's Faces Differ Significantly from Nonpregnant Women.

INTRODUCTION: There is evidence that women's preferences for facial characteristics in men's faces change according to menstrual phase and sexual hormones. Literature indicates that the pregnancy is characterized by a specific sexual hormonal pattern with respect to all other physiological conditions concerning the sexual hormone status during the reproductive age, configuring this physiological condition as an excellent surrogate to study how the sexual hormones may affect many of the aspects concerning the sexual behavior. AIM: The aim of this study was to investigate pregnancy as a model of hormonal influence on women's facial preferences in short-term and long-term relationships and compare the choices of pregnant women with those of nonpregnant women. MAIN OUTCOME MEASURES: Measurement of women's preferences for synthetic men's faces, morphed from hyper-masculine to hypomasculine shape. MATERIALS AND METHODS: Forty-six women in the third trimester of pregnancy, and 70 nonpregnant women took part in the study. All women were shown a composite male face. The sexual dimorphism of the images was enhanced or reduced in a continuous fashion using an open-source morphing program that produced a sequence of 21 pictures of the same face warped from a feminized to a masculinized shape. RESULTS: Pregnant women's choices differed significantly from those of nonpregnant women. In fact, in the context of both a hypothetical short- (M = -0.4 ± 0.11) and long-term relationship (M = -0.4 ± 0.07) pregnant women showed a clear preference for a less masculine man's face than the other group (short-term: M = 0.15 ± 0.13; long-term: M = -0.06 ± 0.15; P < 0.0001). CONCLUSIONS: Women in the third trimester of pregnancy clearly prefer more feminine men's faces, distancing themselves from the choices of women in other physiological conditions concerning the sexual hormonal status during the reproductive age. However, other psychosocial variables may explain this interesting finding.

Comparison of captive lifespan, age-associated liver neoplasias and age-dependent gene expression between two annual fish species: Nothobranchius furzeri and Nothobranchius korthause.

Nothobranchius is a genus of annual fish broadly distributed in South-Eastern Africa and found into temporary ponds generated during the rain seasons and their lifespan is limited by the duration of their habitats. Here we compared two Nothobranchius species from radically different environments: N. furzeri and N. korthausae. We found a large difference in life expectancy (29- against 71-weeks of median life span, 40- against 80-weeks of maximum lifespan, respectively), which correlates with a diverse timing in the onset of several age dependent processes: our data show that N. korthause longer lifespan is associated to retarded onset of age-dependent liver-neoplasia and slower down-regulation of collagen 1 alpha 2 (COL1A2) expression in the skin. On the other hand, the expression of cyclin B1 (CCNB1) in the brain was strongly age-regulated, but with similar profiles in N. furzeri and N. korthausae. In conclusion, our data suggest that the different ageing rate of two species of the same genus could be used as novel tool to investigate and better understand the genetic bases of some general mechanism leading to the complex ageing process, providing a strategy to unravel some of the genetic mechanisms regulating longevity and age-associate pathologies including neoplasias.