Serotonin signaling contribution to an evolutionary success: the jaw joint of vertebrates

Serotonin (5-HT) is an ancient molecule that appeared very early during evolution, and it is present in different phyla. The 5-HT signaling system includes several G-coupled receptors and it is widely conserved in vertebrates. 5-HT is implicated in an astonishing number of biological processes and it has a key role as a morphogen in several complex networks during development before it can act as a neurotransmitter. Recent advances on how serotonin signaling can influence early development and its role in vertebrate morphogenesis come from mice and Xenopus. The emergence of jawed vertebrates (gnathostomes) from jawless vertebrates (agnathans) represents a major event in the evolution of vertebrates. The acquisition of a jaw is assumed to have occurred after the split between gnathostomes and jawless vertebrates. A crucial question concerns what changes were introduced in the developmental patterning programme to obtain a jaw joint that is one of the most innovative inventions in the history of vertebrates. Molecular and developmental studies performed in Xenopus revealed for the first time that serotonin, through the 5-HT2B receptor signaling, is both sufficient and necessary to modulate the shape and functionality of the jaw, including the jaw joint. Accordingly, serotonin can be added to the complex interactive network of extrinsic factors that regulates mandibular arch development, thus contributing to one of the major vertebrate successes in evolution

The age-regulated zinc finger factor ZNF367 is a new modulator of neuroblast proliferation during embryonic neurogenesis.

Global population aging is one of the major social and economic challenges of contemporary society. During aging the progressive decline in physiological functions has serious consequences for all organs including brain. The age-related incidence of neurodegenerative diseases coincides with the sharp decline of the amount and functionality of adult neural stem cells. Recently, we identified a short list of brain age-regulated genes by means of next-generation sequencing. Among them znf367 codes for a transcription factor that represents a central node in gene co-regulation networks during aging, but whose function in the central nervous system (CNS), is completely unknown. As proof of concept, we analysed the role of znf367 during Xenopus laevis neurogenesis. By means of a gene loss of function approach limited to the CNS, we suggested that znf367 might act as a key controller of the neuroblast cell cycle, particularly in the progression of mitosis and spindle checkpoint. A candidate gene approach based on a weighted-gene co-expression network analysis, revealed fancd2 and ska3 as possible targets of znf367. The age-related decline of znf367 correlated well with its role during embryonic neurogenesis, opening new lines of investigation also in adult neurogenesis to improved maintenance and even repair of neuronal function

Social Preference Tests in Zebrafish: A Systematic Review

The use of animal models in biology research continues to be necessary for the development of new technologies and medicines, and therefore crucial for enhancing human and animal health. In this context, the need to ensure the compliance of research with the principles Replacement, Reduction and Refinement (the 3 Rs), which underpin the ethical and human approach to husbandry and experimental design, has become a central issue. The zebrafish (Danio rerio) is becoming a widely used model in the field of behavioral neuroscience. In particular, studying zebrafish social preference, by observing how an individual fish interacts with conspecifics, may offer insights into several neuropsychiatric and neurodevelopmental disorders. The main aim of this review is to summarize principal factors affecting zebrafish behavior during social preference tests. We identified three categories of social research using zebrafish: studies carried out in untreated wild-type zebrafish, in pharmacologically treated wild-type zebrafish, and in genetically engineered fish. We suggest guidelines for standardizing social preference testing in the zebrafish model. The main advances gleaned from zebrafish social behavior testing are discussed, together with the relevance of this method to scientific research, including the study of behavioral disorders in humans. The authors stress the importance of adopting an ethical approach that considers the welfare of animals involved in experimental procedures. Ensuring a high standard of animal welfare is not only good for the animals, but also enhances the quality of our science

Short-Term Effects of Human versus Bovine Sialylated Milk Oligosaccharide Microinjection on Zebrafish Larvae Survival, Locomotor Behavior and Gene Expression

Milk oligosaccharides are a complex class of carbohydrates that act as bioactive factors in numerous defensive and physiological functions, including brain development. Early nutrition can modulate nervous system development and can lead to epigenetic imprinting. We attempted to increase the sialylated oligosaccharide content of zebrafish yolk reserves, with the aim of evaluating any short-term effects of the treatment on mortality, locomotor behavior, and gene expression. Wild-type embryos were microinjected with saline solution or solutions containing sialylated milk oligosaccharides extracted from human and bovine milk. The results suggest that burst activity and larval survival rates were unaffected by the treatments. Locomotion parameters were found to be similar during the light phase between control and treated larvae; in the dark, however, milk oligosaccharide-treated larvae showed increased test plate exploration. Thigmotaxis results did not reveal significant differences in either the light or the dark conditions. The RNA-seq analysis indicated that both treatments exert an antioxidant effect in developing fish. Moreover, sialylated human milk oligosaccharides seemed to increase the expression of genes related to cell cycle control and chromosomal replication, while bovine-derived oligosaccharides caused an increase in the expression of genes involved in synaptogenesis and neuronal signaling. These data shed some light on this poorly explored research field, showing that both human and bovine oligosaccharides support brain proliferation and maturation

Converging Role for REEP1/SPG31 in Oxidative Stress

Mutations in the receptor expression-enhancing protein 1 gene (REEP1) are associated with hereditary spastic paraplegia type 31 (SPG31), a neurological disorder characterized by lengthdependent degeneration of upper motor neuron axons. Mitochondrial dysfunctions have been observed in patients harboring pathogenic variants in REEP1, suggesting a key role of bioenergetics in disease-related manifestations. Nevertheless, the regulation of mitochondrial function in SPG31 remains unclear. To elucidate the pathophysiology underlying REEP1 deficiency, we analyzed in vitro the impact of two different mutations on mitochondrial metabolism. Together with mitochondrial morphology abnormalities, loss-of-REEP1 expression highlighted a reduced ATP production with increased susceptibility to oxidative stress. Furthermore, to translate these findings from in vitro to preclinical models, we knocked down REEP1 in zebrafish. Zebrafish larvae showed a significant defect in motor axon outgrowth leading to motor impairment, mitochondrial dysfunction, and reactive oxygen species accumulation. Protective antioxidant agents such as resveratrol rescued free radical overproduction and ameliorated the SPG31 phenotype both in vitro and in vivo. Together, our findings offer new opportunities to counteract neurodegeneration in SPG31

Loss of ap4s1 in zebrafish leads to neurodevelopmental defects resembling spastic paraplegia 52.

Autosomal recessive spastic paraplegia 52 is caused by biallelic mutations in AP4S1 which encodes a subunit of the adaptor protein complex 4 (AP-4). Using next-generation sequencing, we identified three novel unrelated SPG52 patients from a cohort of patients with cerebral palsy. The discovered variants in AP4S1 lead to reduced AP-4 complex formation in patient-derived fibroblasts. To further understand the role of AP4S1 in neuronal development and homeostasis, we engineered the first zebrafish model of AP-4 deficiency using morpholino-mediated knockdown of ap4s1. In this model, we discovered several phenotypes mimicking SPG52, including altered CNS development, locomotor deficits, and abnormal neuronal excitability

FUNCTIONAL ANALYSIS OF GENES RELATED TO AGING OF THE NERVOUS SYSTEM IN THE PROCESS OF EMBRYONIC NEUROGENESIS

Adult neurogenesis is the process by which new neural cells are generated from a small population of multipotent stem cells located in specific area of the central nervous system (CNS). The age-related incidence of many CNS diseases coincides with a reduced adult neurogenic potential. The regenerative capability and the amount of adult neural stem cells (aNSCs) decline with age contributing to the reduced functionality of the aged brain. Despite the great interest in age related diseases, in Italy alone over-65 people will rise to the 18% value of 2010 to more than 30% in 2050, the molecular factors responsible for age-dependent decay of aNSCs function are almost unknown. The starting point of my work was a list of brain age-regulated genes that has been previously obtained by RNA-Seq and validated by qPCR and in situ hybridization in the aging animal model system Nothobranchius furzeri. Among them, I analysed the expression profile and the function of Mex3A and Znf367 genes, codifying respectively for a RNA binding protein and a transcription factor, in embryonic neurogenesis. Indeed, we envisage that genes controlling age-dependent processes might act in continuity between development, adulthood, and aging. These genes, of unknown function, are expressed in neuroblasts and retinoblasts of zebrafish and Xenopus laevis embryos and in the aNSCs of the short-lived fish N. furzeri. By means of gene gain and loss of function approaches in Xenopus and zebrafish embryos, I obtained preliminary results on the specific function of these genes in regulating the maintenance of a stem cell-like phenotype or differentiated state in the developing CNS. The same genes will be tested to verify their function also in neural stem cells of adult fishes. The identification of genetic mechanisms involved in embryonic and adult neurogenesis represents the first step in defining interventions that can increase neurogenesis in the aged brain and that could lead to improved maintenance and even repair of neuronal function

Ruolo svolto dal recettore serotoninergico 5-HT2B sull'espressione di target diretti dell'acido retinoico durante la morfogenesi oculare di embrioni di Xenopus leavis.

La serotonina (5-HT) è un neuromodulatore che media una grande varietà di funzioni sia nel sistema nervoso centrale che nel sistema nervoso periferico. La 5-HT, inoltre, può agire anche come fattore di crescita e differenziamento durante l'embriogenesi attivando specifici recettori. Nel nostro laboratorio è stato dimostrato che il recettore serotoninergico 5-HT2B ha un ruolo importante nella morfogenesi retinica e craniofacciale in embrioni di Xenopus laevis. Dati presenti in letteratura, hanno suggerito una possibile interazione tra il “signaling” del recettore 5-HT2B e quello mediato dall’acido retinoico (AR), un importante morfogeno embrionale. Nel promotore del recettore 5-HT2B sono presenti elementi di risposta al AR, ed è stato dimostrato che il ‘’signaling’’ viene attivato da AR e da 5-HT2B e che queste molecole agiscono in maniera antagonistica nel differenziamento condrogenico, nello sviluppo dell’ arto e nella regolazione della la proliferazione del mesenchima della “frontonasal mass” del topo (Bhasin et al. 2004; Bhasin et al. 2004). Essendo l’acido retinoico coinvolto anche nel regolare la chiusura della fessura ottica (Matt et al., 2008), durante il mio internato di tesi, ho studiato il ruolo funzionale del recettore 5-HT2B nel mesenchima perioculare durante la morfogenesi dell'occhio, focalizzando la mia attenzione su una possibile interazione tra il “pathway” di segnalazione dell'acido retinoico e quello del recettore 5-HT2B. Usando come sistema modello Xenopus laevis, ho effettuato esperimenti di perdita di funzione genica mediante microiniezione di oligonucleotidi antisenso modificati (morpholini), diretti contro il recettore 2b rilevando insorgenza di coloboma (mancata chiusura della fessura ottica), uno dei più comuni difetti oculari congeniti. Su questi embrioni ho quindi valutato mediante ibridazione in situ e qPCR l’esressione di geni coinvolti nella sintesi e metabolismo dell’acido retinoico come Raldh3, Raldh10 e Dhrs3 e geni noti per essere target diretti dell’acido retinoico nella retina e nel mesenchima perioculare come Pax2, Vax2, Pitx2 e FoxC1. I risultati fino ad ora ottenuti suggeriscono che l'inibizione dell'attività del recettore 5-HT2B possa indurre un aumento dell’attività del AR con conseguente aumento del dominio di espressione dei suoi target diretti. Concludendo, si può ipotizzare, che il “pathway” di segnalazione del recettore 5-HT2B interagisca, giocando un ruolo antagonistico, con il “pathway” di segnalazione dell' AR anche durante la morfogenesi oculare

Oxytocin Receptor Gene Polymorphism in Lactating Dogs

Genetic variations in the oxytocinergic system, known to regulate social behavior throughout the evolution of mammals, are believed to account for differences in mammalian social behavior. Particularly, polymorphic variants of the oxytocin receptor (OXTR) gene have been associated with behavioral variations in both humans and dogs. In this study, we offered evidence of the correlation between levels of salivary oxytocin (sOXT), maternal behavior and a single-nucleotide gene variant in OXTR (rs8679684) in nineteen lactating Labrador Retriever dogs. Carriers of at least one copy of the minor A allele showed higher levels of sOXT and maternal care in comparison with the homozygous T allele carriers. Considering the relevance of mother care in newborn development, these findings could help us to better understand the possible impact of variants in the OXTR gene in selecting dams

Pluripotent Stem Cells as a Preclinical Cellular Model for Studying Hereditary Spastic Paraplegias

Hereditary spastic paraplegias (HSPs) comprise a family of degenerative diseases mostly hitting descending axons of corticospinal neurons. Depending on the gene and mutation involved, the disease could present as a pure form with limb spasticity, or a complex form associated with cerebellar and/or cortical signs such as ataxia, dysarthria, epilepsy, and intellectual disability. The progressive nature of HSPs invariably leads patients to require walking canes or wheelchairs over time. Despite several attempts to ameliorate the life quality of patients that have been tested, current therapeutical approaches are just symptomatic, as no cure is available. Progress in research in the last two decades has identified a vast number of genes involved in HSP etiology, using cellular and animal models generated on purpose. Although unanimously considered invaluable tools for basic research, those systems are rarely predictive for the establishment of a therapeutic approach. The advent of induced pluripotent stem (iPS) cells allowed instead the direct study of morphological and molecular properties of the patient’s affected neurons generated upon in vitro differentiation. In this review, we revisited all the present literature recently published regarding the use of iPS cells to differentiate HSP patient-specific neurons. Most studies have defined patient-derived neurons as a reliable model to faithfully mimic HSP in vitro, discovering original findings through immunological and –omics approaches, and providing a platform to screen novel or repurposed drugs. Thereby, one of the biggest hopes of current HSP research regards the use of patient-derived iPS cells to expand basic knowledge on the disease, while simultaneously establishing new therapeutic treatments for both generalized and personalized approaches in daily medical practice