Adele Leggieri

Ageing is the primary risk factor for neurodegenerative disease, yet experimental models often sacrifice natural age-related disease trajectories for genetically modified models. Teleost fish offer a useful complementary framework in this context. The African turquoise killifish (Nothobranchius furzeri) possesses a naturally compressed lifespan and develops age-associated phenotypes within months, including impaired proteostasis, reduced regenerative capacity, and disease-relevant neurodegenerative signatures. Zebrafish (Danio rerio), on the other hand, age over a longer timeframe, with lifespans that can extend up to ∼5 years under laboratory conditions, but provide exceptional experimental accessibility through well-established and standardised genetic, pharmacological, and behavioural tools for dissecting neural mechanisms in vivo. In the present review, we highlight how these two fish models can be used together to investigate mechanisms linking physiological brain ageing to neurodegenerative vulnerability. We focus on processes like proteostasis collapse, altered translational and nutrient-sensing pathways, and age-related decline in neurogenesis and tissue resilience. We argue that killifish are currently most informative for identifying naturally arising ageing trajectories and early degenerative signatures, whereas zebrafish are particularly suited to causal testing, pathway manipulation, and scalable disease modelling. Rather than treating these systems as alternatives, we propose that they should be considered as complementary vertebrate models. This comparative perspective also highlights how conserved biological processes shape brain ageing and determine vulnerability to age-related neurodegenerative disease.

Williams syndrome (WS) is a multifaceted developmental disorder characterized by a spectrum of physical and intellectual traits. Individuals with WS exhibit friendly, impulsive, and hyper-social behaviours, often coupled with anxiety. WS is caused by a hemizygous microdeletion on chromosome 7q11.23. Among the affected genes, FZD9 is implicated in neural development and synaptic function. Thus, we postulated that disruption of FZD9 might contribute to the behavioural features of WS, and that pharmacological modulation of Wnt signalling, particularly the canonical β-catenin pathway, could offer therapeutic potential. We generated two zebrafish lines carrying predicted loss-of-function alleles of fzd9b , a zebrafish orthologue of FZD9 . Our results demonstrated that mutant fish exhibited altered expression of hypothalamic–pituitary–interrenal axis-related genes, increased neuronal branching, impaired habituation to acoustic startle, and increased anxiety-like behaviour in adulthood. Canonical Wnt/β-catenin pathway gene expression was also altered, together with the expression of other frizzled receptors; however, acute GSK-3 inhibition failed to rescue behavioural phenotypes under the conditions tested. These findings indicate that the selected acute treatment paradigm was insufficient to reverse the phenotype but do not exclude a contribution of canonical Wnt signalling. Expression of fzd9b , wnt5b , and tafa5l was upregulated, further supporting transcriptional dysregulation. Together, our results highlight a conserved role for Fzd9b in modulating stress reactivity and anxiety-like behaviour in zebrafish and support FZD9 as a contributor to WS-associated neurobehavioural alterations.

Mutations in the RBFOX1 gene are associated with psychiatric disorders but how RBFOX1 influences psychiatric disorder vulnerability remains unclear. Recent studies showed that RBFOX proteins mediate the alternative splicing of PAC1, a critical HPA axis activator. Further, RBFOX1 dysfunction is linked to dysregulation of BDNF/TRKB, a pathway promoting neuroplasticity, neuronal survival and stress resilience. Hence, RBFOX1 dysfunction may increase psychiatric disorder vulnerability via HPA axis dysregulation, leading to disrupted development and allostatic overload. To test this hypothesis, we generated a zebrafish rbfox1 loss of function (LoF) line and examined behavioural and molecular effects during development. We found that rbfox1 LoF mutants exhibited hyperactivity, impulsivity and heightened arousal, alongside alterations in proliferation – traits associated with neurodevelopmental and stress-related disorders. In adults, loss of rbfox1 function led to decreased fertility and survival, consistent with allostatic overload. At the molecular level, at larval stages rbfox1 mutants showed increased cortisol levels and disrupted expression of key stress-related genes (bdnf, trkb2, pac1a-hop, crhb, nr3c2). Pharmacological intervention targeting TRKB restored crhb and nr3c2 gene expression and hyperactive and hyperarousal behaviours. In adults, dysregulation of crhb, nr3c2 and bdnf/trkb2 genes was only seen following acute stress exposure. Our findings reveal a fundamental role for RBFOX1 in integrating stress responses through its regulation of BDNF/TRKB and neuroendocrine signalling.

The reproducibility crisis in bioscience, characterized by inconsistent study results, impedes our understanding of biological processes. Global collaborative studies offer a unique solution to this problem. Here, we present a global collaboration using the zebrafish (Danio rerio) novel tank test, a popular behavioral assay for anxiety-like responses. We analyzed data from 20 laboratories worldwide, focusing on housing conditions and experimental setups. Our study included 488 adult zebrafish, tested for 5 min, focusing on a variety of variables. Key findings show that female zebrafish exhibit more anxiety-like behavior than males, highlighting sex as a critical variable. Housing conditions, including higher stocking densities and specific feed types, also influenced anxiety levels. Optimal conditions (5 fish/L) and nutritionally rich feeds (for example, rotifers) mitigated anxiety-like behaviors. Environmental stressors, such as noise and transportation, significantly impacted behavior. We recommend standardizing testing protocols to account for sex differences, optimal stocking densities, nutritionally rich feeds and minimizing stressors to improve the reliability of zebrafish behavioral studies.

The development of effective neurological treatments remains a challenge due to the low permeation across the blood-brain barrier (BBB). The use of nanoparticles for brain drug delivery is promising, but the level of BBB crossing remains low, typically around 0.5-3%. We report the synthesis of negatively charged N-isopropylacrylamide nanogels (diameter 10 nm) displaying in vitro crossing in a human BBB model nearly 5-fold higher than previously reported, and permeating the brain parenchyma of healthy zebrafish with a fully formed BBB. The methylenebisacrylamide crosslinked nanogels, incorporating N-acryloyl-L-proline as pH-responsive unit (electrophoretic mobility = − 0.24 ± 0.05 μm·cm·V-1·s-1), were fluorescently labelled with (4-(2-acryloylaminoethylamino)-7-nitro-2,1,3-benzoxadiazole, to allow in vitro and in vivo tracking. The nanoparticles did not disrupt the in vitro human BBB model up to 0.1 mg/mL, obtained from CD34 positive-derived endothelial cells in co-culture with pericytes, displaying 13.4% permeation at 24 hours; internalization in the BBB endothelial cells was confirmed by confocal microscopy, via clathrin-mediated endocytosis. The ability of nanogels to cross the BBB in vivo without inducing a toxic effect was confirmed in zebrafish, up to 14 days post fertilization.

The reproducibility crisis in bioscience, characterized by inconsistent study results, impedes our understanding of biological processes and global collaborative studies offer a unique solution. This study is the first global collaboration using the zebrafish (Danio rerio) novel tank test, a behavioral assay for anxiety-like responses. We analyzed data from 20 laboratories worldwide, focusing on housing conditions and experimental setups. Our study included 488 adult zebrafish, tested for 5 min, focusing on a variety of variables. Key findings show females exhibit more anxiety-like behavior than males, underscoring sex as a critical variable. Housing conditions, including higher stocking densities and specific feed types, influenced anxiety levels. Optimal conditions (5 fish/L) and nutritionally rich feeds (e.g., rotifers), mitigated anxiety-like behaviors. Environmental stressors, like noise and transportation, significantly impacted behavior. We recommend standardizing protocols to account for sex differences, optimal stocking densities, nutritionally rich feeds, and minimizing stressors to improve zebrafish behavioral study reliability.

Mutations in the RBFOX1 gene are associated with psychiatric disorders but how RBFOX1 influences psychiatric disorder vulnerability remains unclear. Recent studies showed that RBFOX proteins mediate the alternative splicing of PAC1, a critical HPA axis activator. Further, RBFOX1 dysfunction is linked to dysregulation of BDNF/TRKB, a pathway promoting neuroplasticity, neuronal survival, and stress resilience. Hence, RBFOX1 dysfunction may increase psychiatric disorder vulnerability via HPA axis dysregulation, leading to disrupted development and allostatic overload. To test this hypothesis, we generated a zebrafish rbfox1 loss of function (LoF) line and examined behavioural and molecular effects during development. We found that rbfox1 LoF mutants exhibited hyperactivity, impulsivity and heightened arousal, alongside alterations in proliferation – traits associated with neurodevelopmental and stress-related disorders. In adults, loss of rbfox1 function led to decreased fertility and survival, consistent with allostatic overload. At the molecular level, at larval stages rbfox1 mutants showed increased cortisol levels and disrupted expression of key stress-related genes ( bdnf, trkb2, pac1a-hop, crhb, nr3c2 ). Pharmacological intervention targeting TRKB restored crhb and nr3c2 gene expression and hyperactive and hyperarousal behaviours. In adults, dysregulation of crhb, nr3c2 and bdnf/trkb2 genes was only seen following acute stress exposure. Our findings reveal a fundamental role for RBFOX1 in integrating stress responses through its regulation of BDNF/TRKB and neuroendocrine signalling. Abstract Figure:

RBFOX1 is a highly pleiotropic gene that contributes to several psychiatric and neurodevelopmental disorders. Both rare and common variants in RBFOX1 have been associated with several psychiatric conditions, but the mechanisms underlying the pleiotropic effects of RBFOX1 are not yet understood. Here we found that, in zebrafish, rbfox1 is expressed in spinal cord, mid- and hindbrain during developmental stages. In adults, expression is restricted to specific areas of the brain, including telencephalic and diencephalic regions with an important role in receiving and processing sensory information and in directing behaviour. To investigate the contribution of rbfox1 to behaviour, we used rbfox1^sa15940, a zebrafish mutant line with TL background. We found that rbfox1^sa15940 mutants present hyperactivity, thigmotaxis, decreased freezing behaviour and altered social behaviour. We repeated these behavioural tests in a second rbfox1 mutant line with a different genetic background (TU), rbfox1^del19, and found that rbfox1 deficiency affects behaviour similarly in this line, although there were some differences. rbfox1^del19 mutants present similar thigmotaxis, but stronger alterations in social behaviour and lower levels of hyperactivity than rbfox1^sa15940 fish. Taken together, these results suggest that mutations in rbfox1 lead to multiple behavioural changes in zebrafish that might be modulated by environmental, epigenetic and genetic background effects, and that resemble phenotypic alterations present in Rbfox1-deficient mice and in patients with different psychiatric conditions. Our study, thus, highlights the evolutionary conservation of rbfox1 function in behaviour and paves the way to further investigate the mechanisms underlying rbfox1 pleiotropy on the onset of neurodevelopmental and psychiatric disorders.

Collagens are the most abundant proteins in vertebrates and constitute the major components of the extracellular matrix. Collagens play an important and multifaceted role in the development and functioning of the nervous system and undergo structural remodeling and quantitative modifications during aging. Here, we investigated the age-dependent regulation of col4a1 and col25a1 in the brain of the short-lived vertebrate Nothobranchius furzeri, a powerful model organism for aging research due to its natural fast-aging process and further characterized typical hallmarks of brain aging in this species. We showed that col4a1 and col25a1 are relatively well conserved during vertebrate evolution, and their expression significantly increases in the brain of N. furzeri upon aging. Noteworthy, we report that both col4a1 and col25a1 are expressed in cells with a neuronal phenotype, unlike what has already been documented in mammalian brain, in which only col25a1 is considered a neuronal marker, whereas col4a1 seems to be expressed only in endothelial cells. Overall, our findings encourage further investigation on the role of col4a1 and col25a1 in the biology of the vertebrate brain as well as the onset of aging and neurodegenerative diseases.