Single-cell sequencing unveils the impact of aging on the progenitor cell diversity in the telencephalon of the female killifish N. furzeri.

The African turquoise killifish (Nothobranchius furzeri) combines a short lifespan with spontaneous age-associated loss of neuro-regenerative capacity, an intriguing trait atypical for a teleost. The impact of aging on the cellular composition of the adult stem cell niches, leading to this dramatic decline in the postnatal neuro- and gliogenesis, remains elusive. Single-cell RNA sequencing of the telencephalon of young adult female killifish of the short-lived GRZ-AD strain unveiled progenitors of glial and non-glial nature, different excitatory and inhibitory neuron subtypes, as well as non-neural cell types. Sub-clustering of the progenitors identified four radial glia (RG) cell types, two non-glial progenitor (NGP) and four intermediate (intercell) cell states. Two astroglia-like, one ependymal, and one neuroepithelial-like (NE) RG subtype were found at different locations in the forebrain in line with their role, while proliferative, active NGPs were spread throughout. Lineage inference pointed to NE-RG and NGPs as start and intercessor populations for glio- and neurogenesis. Upon aging, single-cell RNA sequencing revealed major perturbations in the proportions of the astroglia and intercell states, and in the molecular signatures of specific subtypes, including altered MAPK, mTOR, Notch, and Wnt pathways. This cell catalog of the young regeneration-competent killifish telencephalon, combined with the evidence for aging-related transcriptomic changes, presents a useful resource to understand the molecular basis of age-dependent neuroplasticity. This data is also available through an online database (killifishbrain_scseq).

A short dasatinib and quercetin treatment is sufficient to reinstate potent adult neuroregenesis in the aged killifish.

The young African turquoise killifish has a high regenerative capacity, but loses it with advancing age, adopting several aspects of the limited form of mammalian regeneration. We deployed a proteomic strategy to identify pathways that underpin the loss of regenerative power caused by aging. Cellular senescence stood out as a potential brake on successful neurorepair. We applied the senolytic cocktail Dasatinib and Quercetin (D + Q) to test clearance of chronic senescent cells from the aged killifish central nervous system (CNS) as well as rebooting the neurogenic output. Our results show that the entire aged killifish telencephalon holds a very high senescent cell burden, including the parenchyma and the neurogenic niches, which could be diminished by a short-term, late-onset D + Q treatment. Reactive proliferation of non-glial progenitors increased substantially and lead to restorative neurogenesis after traumatic brain injury. Our results provide a cellular mechanism for age-related regeneration resilience and a proof-of-concept of a potential therapy to revive the neurogenic potential in an already aged or diseased CNS.

Molecular and Biochemical Analyses of Aging Hallmarks in the Central Nervous System of the Fast-Aging African Turquoise Killifish.

As modern society is graying, aging research and biogerontology models, in which the aging process can be studied, are becoming increasingly important. A proper aging model can be defined as one that displays many of the aging hallmarks. Here, we provide two different practical approaches-namely, real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting-that can be used to investigate cellular senescence (RT-qPCR for p21 and p27), altered intercellular communication/inflammaging (RT-qPCR for il-10, sirt-1, il-6, il-1b, il-8, and tnf), and oxidative stress (western blotting for 4-HNE) in the killifish central nervous system, and, more specifically, in the retina, optic tectum, and telencephalon. These molecular and biochemical analyses are a first step in confirming the aging characteristics but should preferably be combined with morphological analyses.

Age-related alterations in the behavioral response to a novel environment in the African turquoise killifish (Nothobranchius furzeri).

The African turquoise killifish (Nothobranchius furzeri) has emerged as a popular model organism for neuroscience research in the last decade. One of the reasons for its popularity is its short lifespan for a vertebrate organism. However, little research has been carried out using killifish in behavioral tests, especially looking at changes in their behavior upon aging. Therefore, we used the open field and the novel tank diving test to unravel killifish locomotion, exploration-related behavior, and behavioral changes over their adult lifespan. The characterization of this behavioral baseline is important for future experiments involving pharmacology to improve the aging phenotype. In this study, two cohorts of fish were used, one cohort was tested in the open field test and one cohort was tested in the novel tank diving test. Each cohort was tested from the age of 6 weeks to the age of 24 weeks and measurements were performed every three weeks. In the open field test, we found an increase in the time spent in the center zone from 18 weeks onward, which could indicate altered exploration behavior. However, upon aging, the fish also showed an increased immobility frequency and duration. In addition, after the age of 15 weeks, their locomotion decreased. In the novel tank diving test, we did not observe this aging effect on locomotion or exploration. Killifish spent around 80% of their time in the bottom half of the tank, and we could not observe habituation effects, indicating slow habituation to novel environments. Moreover, we observed that killifish showed homebase behavior in both tests. These homebases are mostly located near the edges of the open field test and at the bottom of the novel tank diving test. Altogether, in the open field test, the largest impact of aging on locomotion and exploration was observed beyond the age of 15 weeks. In the novel tank diving test, no effect of age was found. Therefore, to test the effects of pharmacology on innate behavior, the novel tank diving test is ideally suited because there is no confounding effect of aging.

Prothoracicostatic Activity of the Ecdysis-Regulating Neuropeptide Crustacean Cardioactive Peptide (CCAP) in the Desert Locust.

Accurate control of innate behaviors associated with developmental transitions requires functional integration of hormonal and neural signals. Insect molting is regulated by a set of neuropeptides, which trigger periodic pulses in ecdysteroid hormone titers and coordinate shedding of the old cuticle during ecdysis. In the current study, we demonstrate that crustacean cardioactive peptide (CCAP), a structurally conserved neuropeptide described to induce the ecdysis motor program, also exhibits a previously unknown prothoracicostatic activity to regulate ecdysteroid production in the desert locust, Schistocerca gregaria. We identified the locust genes encoding the CCAP precursor and three G protein-coupled receptors that are activated by CCAP with EC50 values in the (sub)nanomolar range. Spatiotemporal expression profiles of the receptors revealed expression in the prothoracic glands, the endocrine organs where ecdysteroidogenesis occurs. RNAi-mediated knockdown of CCAP precursor or receptors resulted in significantly elevated transcript levels of several Halloween genes, which encode ecdysteroid biosynthesis enzymes, and in elevated ecdysteroid levels one day prior to ecdysis. Moreover, prothoracic gland explants exhibited decreased secretion of ecdysteroids in the presence of CCAP. Our results unequivocally identify CCAP as the first prothoracicostatic peptide discovered in a hemimetabolan species and reveal the existence of an intricate interplay between CCAP signaling and ecdysteroidogenesis.

Aging impairs the essential contributions of non-glial progenitors to neurorepair in the dorsal telencephalon of the Killifish Nothobranchius furzeri.

The aging central nervous system (CNS) of mammals displays progressive limited regenerative abilities. Recovery after loss of neurons is extremely restricted in the aged brain. Many research models fall short in recapitulating mammalian aging hallmarks or have an impractically long lifespan. We established a traumatic brain injury model in the African turquoise killifish (Nothobranchius furzeri), a regeneration-competent vertebrate that evolved to naturally age extremely fast. Stab-wound injury of the aged killifish dorsal telencephalon unveils an impaired and incomplete regeneration response when compared to young individuals. In the young adult killifish, brain regeneration is mainly supported by atypical non-glial progenitors, yet their proliferation capacity clearly declines with age. We identified a high inflammatory response and glial scarring to also underlie the hampered generation of new neurons in aged fish. These primary results will pave the way to unravel the factor age in relation to neurorepair, and to improve therapeutic strategies to restore the injured and/or diseased aged mammalian CNS.

Modeling Neuroregeneration and Neurorepair in an Aging Context: The Power of a Teleost Model.

Aging increases the risk for neurodegenerative disease and brain trauma, both leading to irreversible and multifaceted deficits that impose a clear societal and economic burden onto the growing world population. Despite tremendous research efforts, there are still no treatments available that can fully restore brain function, which would imply neuroregeneration. In the adult mammalian brain, neuroregeneration is naturally limited, even more so in an aging context. In view of the significant influence of aging on (late-onset) neurological disease, it is a critical factor in future research. This review discusses the use of a non-standard gerontology model, the teleost brain, for studying the impact of aging on neurorepair. Teleost fish share a vertebrate physiology with mammals, including mammalian-like aging, but in contrast to mammals have a high capacity for regeneration. Moreover, access to large mutagenesis screens empowers these teleost species to fill the gap between established invertebrate and rodent models. As such, we here highlight opportunities to decode the factor age in relation to neurorepair, and we propose the use of teleost fish, and in particular killifish, to fuel new research in the neuro-gerontology field.