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Inhibitors of DNA (Id) are key transcription factors (TFs) regulating neurogenic processes. They belong to the helix-loop-helix (HLH) TF family and are dominant negative regulators of basic HLH proteins (bHLHs). Specifically, they inhibit cell differentiation and enhance cell proliferation and motility. The Id family includes four members, Id1, Id2, Id3, and Id4, which have been identified in nearly all vertebrates. The transcript catalog of the African turquoise killifish, Nothobranchius furzeri, contains all four TFs and has evolved showing positive selection for Id3. N. furzeri, a teleost, is the short-lived vertebrate and is gaining increasing scientific interest as a new model organism in aging research. It is characterized by embryonic diapause, explosive sexual maturation, and rapid aging. In this study, we investigated both the expression and the role of Id3 in the brain of this model organism. Interestingly, Id3 was upregulated age-dependently along with a distribution pattern resembling that of other vertebrates. Additionally, the gene has undergone positive selection during evolution and shows a high degree of conservation relative to that of other vertebrates. These features make N. furzeri a valid tool for aging studies and a potential model in translational research.

Nesfatin-1 (Nesf-1) was identified as an anorexigenic and well conserved molecule in rodents and fish. While tissue distribution of NUCB2 (Nucleobindin 2)/Nesf-1 is discretely known in vertebrates, reports on ontogenetic expression are scarce. Here, we examine the age-related central and peripheral expression of NUCB2/Nesf-1 in the teleost African turquoise killifish Nothobranchius furzeri, a consolidated model organism for aging research. We focused our analysis on brain areas responsible for the regulation of food intake and the rostral intestinal bulb, which is analogous of the mammalian stomach. We hypothesize that in our model, the stomach equivalent structure is the main source of NUCB2 mRNA, displaying higher expression levels than those observed in the brain, mainly during aging. Remarkably, its expression significantly increased in the rostral intestinal bulb compared to the brain, which is likely due to the typical anorexia of aging. When analyzing the pattern of expression, we confirmed the distribution in diencephalic areas involved in food intake regulation at all age stages. Interestingly, in the rostral bulb, NUCB2 mRNA was localized in the lining epithelium of young and old animals, while Nesf-1 immunoreactive cells were distributed in the submucosae. Taken together, our results represent a useful basis for gaining deeper knowledge regarding the mechanisms that regulate food intake during vertebrate aging.

Neurotrophins contribute to the complexity of vertebrate nervous system, being involved in cognition and memory. Abnormalities associated with neurotrophin synthesis may lead to neuropathies, neurodegenerative disorders and age-associated cognitive decline. The genome of teleost fishes contains homologs of some mammalian neurotrophins as well as a gene coding for an additional neurotrophin (NT-6). In this study, we characterized this specific neurotrophin in the short-lived fish Nothobranchius furzeri, a relatively new model for aging studies. Thus, we report herein for the first time the age-related expression of a neurotrophin in a non-mammalian vertebrate. Interestingly, we found comparable expression levels of NT-6 in the brain of both young and old animals. More in detail, we used a locked nucleic acid probe and a riboprobe to investigate the neuroanatomical distribution of NT-6 mRNA revealing a significant expression of the neurotrophin in neurons of the forebrain (olfactory bulbs, dorsal and ventral telencephalon, and several diencephalic nuclei), midbrain (optic tectum, longitudinal tori, and semicircular tori), and hindbrain (valvula and body of cerebellum, reticular formation and octavolateral area of medulla oblongata). By combining in situ hybridization and immunohistochemistry, we showed that NT-6 mRNA is synthesized in mature neurons. These results contribute to better understanding the evolutionary history of neurotrophins in vertebrates, and their role in the adult brain.

Nerve growth factor (NGF) receptors are evolutionary conserved molecules, and in mammals are considered necessary for ensuring the survival of cholinergic neurons. The age-dependent regulation of NTRK1/NTRKA and p75/NGFR in mammalian brain results in a reduced response of the cholinergic neurons to neurotrophic factors and is thought to play a role in the pathogenesis of neurodegenerative diseases. Here, we study the age-dependent expression of NGF receptors (NTRK1/NTRKA and p75/NGFR) in the brain of the short-lived teleost fish Nothobranchius furzeri. We observed that NTRK1/NTRKA is more expressed than p75/NGFR in young and old animals, although both receptors do not show a significant age-dependent change. We then study the neuroanatomical organization of the cholinergic system, observing that cholinergic fibers project over the entire neuroaxis while cholinergic neurons appear restricted to few nuclei situated in the equivalent of mammalian subpallium, preoptic area and rostral reticular formation. Finally, our experiments do not confirm that NTRK1/NTRKA and p75/NGFR are expressed in cholinergic neuronal populations in the adult brain of N. furzeri. To our knowledge, this is the first study where NGF receptors have been analyzed in relation to the cholinergic system in a fish species along with their age-dependent modulation. We observed differences between mammals and fish, which make the African turquoise killifish an attractive model to further investigate the fish specific NGF receptors regulation.