Eva Terzibasi Tozzini

The short-lived annual fish Nothobranchius furzeri shows a typical teleost aging process reinforced by high incidence of age-dependent neoplasias.

The annual fish Nothobranchius furzeri is the shortest-lived vertebrate which can be cultured in captivity. Here, we performed a histopathological analysis of age-related lesions in this species. Post-mortem analysis revealed lesions in liver (~90%), kidney (~75%), heart (~70%) and gonads (~40%) which are similar to those previously described in the small teleost Poecilia reticulata. In addition, a high incidence of neoplasias was observed in liver (~35%) and kidney (~25%). Different laboratory strains of N. furzeri show large genetic differences in longevity. Cross-sectional analysis revealed a clear age-dependent increase in the incidence of liver neoplasias which was accelerated in a short-lived strain. Cross-sectional analysis of gonads revealed sex-specific differences in the occurrence of lesions, with males being more severely affected than females. In conclusion, our analysis demonstrates that short life span in N. furzeri is a consequence of a typical teleost aging process which determines systemic failure of homeostasis functions rather than of a single organ or apparatus. Unlike other teleosts, however, this scenario is reinforced by high incidence of age-dependent neoplasias, making this species a promising model to analyze the molecular pathways of age-dependent spontaneous tumorigenesis.

Adult neurogenesis in the short‐lived teleost Nothobranchius furzeri: localization of neurogenic niches, molecular characterization and effects of aging

We studied adult neurogenesis in the short‐lived annual fish Nothobranchius furzeri and quantified the effects of aging on the mitotic activity of the neuronal progenitors and the expression of glial fibrillary acid protein (GFAP) in the radial glia. The distribution of neurogenic niches is substantially similar to that of zebrafish and adult stem cells generate neurons, which persist in the adult brain. As opposed to zebrafish, however, the N. furzeri genome contains a doublecortin (DCX) gene. Doublecortin is transiently expressed by newly generated neurons in the telencephalon and optic tectum (OT). We also analyzed the expression of the microRNA miR‐9 and miR‐124 and found that they have complementary expression domains: miR‐9 is expressed in the neurogenic niches of the telencephalon and the radial glia of the OT, while miR‐124 is expressed in differentiated neurons. The main finding of this paper is the demonstration of an age‐dependent decay in adult neurogenesis. Using unbiased stereological estimates of cell numbers, we detected an almost fivefold decrease in the number of mitotically active cells in the OT between young and old age. This reduced mitotic activity is paralleled by a reduction in DCX labeling. Finally, we detected a dramatic up‐regulation of GFAP in the radial glia of the aged brain. This up‐regulation is not paralleled by a similar up‐regulation of S100B and Musashi‐1, two other markers of the radial glia. In summary, the brain of N. furzeri replicates two typical hallmarks of mammalian aging: gliosis and reduced adult neurogenesis.

Parallel evolution of senescence in annual fishes in response to extrinsic mortality

BackgroundEarly evolutionary theories of aging predict that populations which experience low extrinsic mortality evolve a retarded onset of senescence. Experimental support for this theory in vertebrates is scarce, in part for the difficulty of quantifying extrinsic mortality and its condition- and density-dependent components that –when considered- can lead to predictions markedly different to those of the “classical” theories. Here, we study annual fish of the genus Nothobranchius whose maximum lifespan is dictated by the duration of the water bodies they inhabit. Different populations of annual fish do not experience different strengths of extrinsic mortality throughout their life span, but are subject to differential timing (and predictability) of a sudden habitat cessation. In this respect, our study allows testing how aging evolves in natural environments when populations vary in the prospect of survival, but condition-dependent survival has a limited effect. We use 10 Nothobranchius populations from seasonal pools that differ in their duration to test how this parameter affects longevity and aging in two independent clades of these annual fishes.ResultsWe found that replicated populations from a dry region showed markedly shorter captive lifespan than populations from a humid region. Shorter lifespan correlated with accelerated accumulation of lipofuscin (an established age marker) in both clades. Analysis of wild individuals confirmed that fish from drier habitats accumulate lipofuscin faster also under natural conditions. This indicates faster physiological deterioration in shorter-lived populations.ConclusionsOur data provide a strong quantitative example of how extrinsic mortality can shape evolution of senescence in a vertebrate clade. Nothobranchius is emerging as a genomic model species. The characterization of pairs of closely related species with different longevities should provide a powerful paradigm for the identification of genetic variations responsible for evolution of senescence in natural populations.

Repeated intraspecific divergence in life span and aging of African annual fishes along an aridity gradient.

Life span and aging are substantially modified by natural selection. Across species, higher extrinsic (environmentally related) mortality (and hence shorter life expectancy) selects for the evolution of more rapid aging. However, among populations within species, high extrinsic mortality can lead to extended life span and slower aging as a consequence of condition‐dependent survival. Using within‐species contrasts of eight natural populations of Nothobranchius fishes in common garden experiments, we demonstrate that populations originating from dry regions (with short life expectancy) had shorter intrinsic life spans and a greater increase in mortality with age, more pronounced cellular and physiological deterioration (oxidative damage, tumor load), and a faster decline in fertility than populations from wetter regions. This parallel intraspecific divergence in life span and aging was not associated with divergence in early life history (rapid growth, maturation) or pace‐of‐life syndrome (high metabolic rates, active behavior). Variability across four study species suggests that a combination of different aging and life‐history traits conformed with or contradicted the predictions for each species. These findings demonstrate that variation in life span and functional decline among natural populations are linked, genetically underpinned, and can evolve relatively rapidly.

Neurotrophin Trk receptors in the brain of a teleost fish, Nothobranchius furzeri

Trk neurotrophin receptors are transmembrane tyrosine kinase proteins known as TrkA, TrkB, and TrkC. TrkA is the high affinity receptor for nerve growth factor, TrkB is the one for both brain‐derived neurotrophic factor and neurotrophin‐4, and TrkC is the preferred receptor for neurotrophin‐3. In the adult mammalian brain, neurotrophins are important regulators of neuronal function and plasticity. This study is based on Nothobranchius furzeri, a teleost fish that is becoming an ideal candidate as animal model for aging studies because its life expectancy in captivity is of just 3 months. In adult N. furzeri, all three investigated neurotrophin Trk receptors were immunohistochemically detected in each brain region. TrkA positive neuronal perikarya were localized in the dorsal and ventral areas of the telencephalon and in the cortical nucleus; TrkB immunoreactivity was observed in neuronal perikarya of the dorsal and ventral areas of the telencephalon, the diffuse inferior lobe of the hypothalamus, and Purkinje cells; TrkC positive neuronal perikarya were detected in the most aboral region of the telencephalon, in the magnocellular preoptic nucleus and in few neurons dispersed in the hypothalamus. Numerous positive fibers were widely distributed throughout the brain. Radial glial cells lining the mesencephalic and rhombencephalic ventricles showed immunoreactivity to all three Trks. These findings suggest an involvement of neurotrophins in many aspects of biology of adult N. furzeri. Microsc. Res. Tech., 2012.

Immunolocalization of S100-like protein in the brain of an emerging model organism: Nothobranchius furzeri.

The S100 protein in nervous tissue appears to play important roles in regulating neuronal differentiation, glial proliferation, plasticity, development, axonal growth, and in neurogenetic processes. In fish, the adult neurogenic activity is much higher than in mammals. In this study, the localization of S100 protein was investigated in the brain of annual teleost fish, Nothobranchius furzeri, which is an emerging model organism for aging research. By immunohistochemical techniques, S100 immunoreactivity (IR) was detected in glial cells, small neurons, and fibers throughout all regions of central nervous system (CNS) with different pattern of distribution. In the telencephalon, S100 IR was seen in the olphactory bulbs and in different areas of the telencephalic hemispheres. In the diencephalon, S100 positivity was observed in the habenular nuclei of the epithalamus, in the cortical thalamic nucleus, in the dorsal, ventral and caudal portions, the latter with the posterior recessus nucleus, and in the diffuse inferior lobe of the hypothalamus, along the diencephalic ventricle and in the dorsal optic tract. In the mesencephalon, S100 IR was observed in the longitudinal tori, in the optic tectum, and along the mesencephalic ventricle. In the rhombencephalon, S100 IR was shown in valvula and body of the cerebellum, and in some nuclei of the medulla oblongata. The results suggest that S100 may play a key role in the maintenance of the CNS and in neurogenesis processes in the adulthood.

Regulation of microRNA expression in the neuronal stem cell niches during aging of the short-lived annual fish Nothobranchius furzeri

In the last decade, our group has intensively studied the annual fish Nothobranchius furzeri as a new experimental model in Biology specifically applied to aging research. We previously studied adult neuronal stem cells of N. furzeri in vivo and we demonstrated an age-dependent decay in adult neurogenesis. More recently we identified and quantified the expression of miRNAs in the brain of N. furzeri and we detected 165 conserved miRNAs and found that brain aging in this fish is associated with coherent up-regulation of well-known tumor suppressor miRNAs, as well as down-regulation of well-known onco miRNAs~– In the present work we characterized the expression of miR-15a, miR-20a, and microRNA cluster 17–92 in the principal neurogenic niches of the brain of young and old subjects of N. furzeri, by using in situ hybridization techniques, together with proliferating-cell nuclear antigen immuno-staining for a simultaneous visualization of the neuronal progenitors. We found that: (1) the expression of miR-15a is higher in the brain of old subjects and concentrates mainly in the principal neurogenic niches of telencephalon and optic tectum, (2) the expression of miR-20a is higher in the brain of young subjects, but more widespread to the areas surrounding the neurogenic niches, (3) finally, the expression of the microRNA cluster 17–92 is higher in the brain of young subjects, concentrated mainly in the principal neurogenic niches of telencephalon and cerebellum, and with reduced intensity in the optic tectum. Taken together, our data show that these microRNAs, originally identified in whole-brain analysis, are specifically regulated in the stem cell niche during aging.

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.