Study of brain aging and life extension in a short-lived vertebrate

La presente ricerca è stata incentrata sulla validazione dell’uso del pesce annuale Africano Nothobranchius furzeri come nuovo modello vertebrato per gli studi sull’invecchiamento, e nella sua applicazione in specifiche manipolazioni sperimentali in grado di modulare l’aspettativa di vita e la manifestazione dei tipici processi connessi al fenotipo senescente. A tale scopo abbiamo dimostrato che l’abbassamento della temperatura nell’ambiente di allevamento e la somministrazione per via orale di Resveratrolo (sostanza di origine vegetale nota per le sue proprietà antiossidanti) sono in grado di allungare significativamente l’aspettativa di vita e prevenire tanto il deterioramento istologico, quanto il declino cognitivo e la riduzione dell’efficienza motoria età-dipendente del nostro modello. Infine abbiamo dimostrato l’esistenza in natura di popolazioni distinte (“isolati”) di N. furzeri con diversa aspettativa di vita, da cui poter trarre informazioni importanti per successivi studi di genetica quantitativa, volti a identificare geni o gruppi di essi direttamente coinvolti nella modulazione dell’ aspettativa di vita del nostro modello vertebrat

Age-Dependent Regulation of Notch Family Members in the Neuronal Stem Cell Niches of the Short-Lived Killifish Nothobranchius furzeri

Background: The annual killifish Nothobranchius furzeri is a new experimental model organism in biology, since it represents the vertebrate species with the shortest captive life span and also shows the fastest maturation and senescence recorded in the laboratory. Here, we use this model to investigate the age-dependent decay of neurogenesis in the telencephalon (brain region sharing the same embryonic origin with the mammalian adult niches), focusing on the expression of the Notch pathway genes. Results: We observed that the major ligands/receptors of the pathway showed a negative correlation with age, indicating age-dependent downregulation of the Notch pathway. Moreover, expression of notch1a was clearly limited to active neurogenic niches and declined during aging, without changing its regional patterning. Expression of notch3 is not visibly influenced by aging. Conclusion: Both expression pattern and regulation differ between notch1a and notch3, with the former being limited to mitotically active regions and reduced by aging and the latter being present in all cells with a neurogenic potential, regardless of the level of their actual mitotic activity, and so is less influenced by age. This finally suggests a possible differential role of the two receptors in the regulation of the niche proliferative potential throughout the entire fish life

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

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 (S100β) 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

Quantification of noradrenergic-, dopaminergic-, and tectal-neurons during aging in the short-lived killifish Nothobranchius furzeri

Parkinson's disease (PD) is characterized by phosphorylation and aggregation of the protein alpha-Synuclein and ensuing neuronal death progressing from the noradrenergic locus coeruleus to midbrain dopaminergic neurons. In 2019, Matsui and colleagues reported a spontaneous age-dependent degeneration of dopaminergic neurons and an even greater neurodegeneration of the noradrenergic neurons in the short-lived killifish Nothobranchius furzeri. Given the great possible relevance of a spontaneous model for PD, we assessed neurodegeneration of noradrenergic and dopaminergic neurons in two further laboratory strains of N. furzeri. We implemented, for the first time in N. furzeri, a whole-brain clarification technique and proceeded to entire 3D nuclei reconstruction to quantify total cell numbers in two different stains of N. furzeri. In both strains, we observed that age-dependent neurodegeneration is limited to the locus coeruleus and does not involve the posterior tuberculum. We also applied 3D counting to the optic tectum, an area of active adult neurogenesis, and detected an increase of neurons with age. Our results confirm age-dependent neurodegeneration of noradrenergic neurons, a condition reminiscent of the presymptomatic stage of PD indicating that N. furzeri could be used in the future to identify modifying factors for age-dependent neurodegeneration and open the intriguing possibility that natural genetic variation may influence the susceptibility of dopaminergic neurons

Large Differences in Aging Phenotype between Strains of the Short-Lived Annual Fish Nothobranchius furzeri

BACKGROUND: A laboratory inbred strain of the annual fish Nothobranchius furzeri shows exceptionally short life expectancy and accelerated expression of age markers. In this study, we analyze new wild-derived lines of this short-lived species. METHODOLOGY/PRINCIPAL FINDINGS: We characterized captive survival and age-related traits in F1 and F2 offspring of wild-caught N. furzeri. Wild-derived N. furzeri lines showed expression of lipofuscin and neurodegeneration at age 21 weeks. Median lifespan in the laboratory varied from to 20 to 23 weeks and maximum lifespan from 25 to 32 weeks. These data demonstrate that rapid age-dependent decline and short lifespan are natural characteristics of this species. The N. furzeri distribution range overlaps with gradients in altitude and aridity. Fish from more arid habitats are expected to experience a shorter survival window in the wild. We tested whether captive lines stemming from semi-arid and sub-humid habitats differ in longevity and expression of age-related traits. We detected a clear difference in age-dependent cognitive decline and a slight difference in lifespan (16% for median, 15% for maximum lifespan) between these lines. Finally, we observed shorter lifespan and accelerated expression of age-related markers in the inbred laboratory strain compared to these wild-derived lines. CONCLUSIONS/SIGNIFICANCE: Owing to large differences in aging phenotypes in different lines, N. furzeri could represent a model system for studying the genetic control of life-history traits in natural populations

Age-related central regulation of orexin and NPY in the short-lived African killifish Nothobranchius furzeri

Orexin A (OXA) and neuropeptide Y (NPY) are two hypothalamic neuropeptides involved in the regulation of feeding behavior and food intake in all vertebrates. Accumulating evidences document that they undergo age-related modifications, with consequences on metabolism, sleep/wake disorders and progression of neurodegenerations. The present study addressed the age related changes in expression and distribution of orexin A (its precursor is also known as hypocretin\u2014HCRT) and NPY, and their regulation by food intake in the short-lived vertebrate model Nothobranchius furzeri. Our experiments, conducted on male specimens, show that: (a) HCRT and OXA and NPY mRNA and protein are localized in neurons of diencephalon and optic tectum, as well as in numerous fibers projecting through the entire neuroaxis, and are colocalized in specific nuclei; (b) in course of aging, HCRT and NPY expressing neurons are localized also in telencephalon and rhombencephalon; (c) HCRT expressing neurons increased slightly in the diencephalic area of old animals and in fasted animals, whereas NPY increased sharply; (d) central HCRT levels are not regulated neither in course of aging nor by food intake; and (e) central NPY levels are augmented in course of aging, and regulated by food intake only in young. These findings represent a great novelty in the study of central orexinergic and NPY-ergic systems in vertebrates', demonstrating an uncommon and unprecedented described regulation of these two orexigenic neuropeptides

Effect Of Priming On Seed Vigor Of Wheat (Triticum Aestivum L.)

Priming is a process that controls the process of hydration of seeds for the ongoing metabolic processes before germination. Research on priming was conducted at ICERI seed laboratory from May to September 2009 to evaluate the effect of different priming methods on wheat seed vigor. Physical properties and chemical composition of seed were evaluated before seeds were treated. The priming treatment were conducted by soaking 250 g of seed in 500 mL of solution for hydropriming and halopriming. Two seed lots of Nias and Dewata variety were subjected to heated and unheated distilled water for 12hours and subjected to KCl and CaCl2 at 10, 20, and 30 ppm and unprimed seed. The experiment were arranged in completely randomized design, replicated thrice. Vigor evaluation by observed seed germination, simultaneity growth, germination rate, seedling dry weight, electric conductivity of seed leakage and length of primary root. The results showed that highest germination, simultaneity growth, seedling dry weight, and length of primary root, were priming treatment with KCl 30 ppm and CaCl2 20 and 30 ppm. Priming with distilled water for 12 hours gave higher germination percentage and simultaneity growth

Gender Separation Increases Somatic Growth in Females but Does Not Affect Lifespan in Nothobranchius furzeri

According to life history theory, physiological and ecological traits and parameters influence an individual's life history and thus, ultimately, its lifespan. Mating and reproduction are costly activities, and in a variety of model organisms, a negative correlation of longevity and reproductive effort has been demonstrated. We are employing the annual killifish Nothobranchius furzeri as a vertebrate model for ageing. N. furzeri is the vertebrate displaying the shortest known lifespan in captivity with particular strains living only three to four months under optimal laboratory conditions. The animals show explosive growth, early sexual maturation and age-dependent physiological and behavioural decline. Here, we have used N. furzeri to investigate a potential reproduction-longevity trade-off in both sexes by means of gender separation. Though female reproductive effort and offspring investment were significantly reduced after separation, as investigated by analysis of clutch size, eggs in the ovaries and ovary mass, the energetic surplus was not reallocated towards somatic maintenance. In fact, a significant extension of lifespan could not be observed in either sex. This is despite the fact that separated females, but not males, grew significantly larger and heavier than the respective controls. Therefore, it remains elusive whether lifespan of an annual species evolved in periodically vanishing habitats can be prolonged on the cost of reproduction at all