Platelet derived growth factor B gene expression in the Xenopus laevis developing central nervous system

Platelet-derived growth factor B (PDGF-B) belongs to the mitogen and growth factor family and like the other members it has many roles in cell differentiation, proliferation and migration during development, adult life and in pathological conditions. Among them it has been observed that aberrant PDGF signalling is frequently linked to glioma development and progression, and Pdgf-b over-expression in mouse neural progenitors leads to the formation of gliomas. Despite this evidence, the mechanisms underlying PDGF-B driven tumorigenesis and its role during brain development are not fully understood. In order to contribute to clarifying possible new roles of pdgf-b signalling, we present here the embryonic gene expression pattern of pdgf-b, so far unknown in early vertebrate development. By using Xenopus laevis as a model system we performed qRT-PCR and whole mount in situ hybridization. Pdgf-b mRNA is expressed in discrete regions of the developing central nervous system, in the cranial nerve placodes and in the notochord. We also compared the gene expression of pdgf-b with that of its receptor pdgfr-a suggesting so far unsuspected roles for this signalling pathway during the development of specific embryonic structures

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

The Stemness Gene Mex3A Is a Key Regulator of Neuroblast Proliferation During Neurogenesis

Mex3A is an RNA binding protein that can also act as an E3 ubiquitin ligase to control gene expression at the post-transcriptional level. In intestinal adult stem cells, MEX3A is required for cell self-renewal and when overexpressed, MEX3A can contribute to support the proliferation of different cancer cell types. In a completely different context, we found mex3A among the genes expressed in neurogenic niches of the embryonic and adult fish brain and, notably, its expression was downregulated during brain aging. The role of mex3A during embryonic and adult neurogenesis in tetrapods is still unknown. Here, we showed that mex3A is expressed in the proliferative region of the developing brain in both Xenopus and mouse embryos. Using gain and loss of gene function approaches, we showed that, in Xenopus embryos, mex3A is required for neuroblast proliferation and its depletion reduced the neuroblast pool, leading to microcephaly. The tissue-specific overexpression of mex3A in the developing neural plate enhanced the expression of sox2 and msi-1 keeping neuroblasts into a proliferative state. It is now clear that the stemness property of mex3A, already demonstrated in adult intestinal stem cells and cancer cells, is a key feature of mex3a also in developing brain, opening new lines of investigation to better understand its role during brain aging and brain cancer development

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

Studio del ruolo del fattore PDGF-B in fenomeni di chemoattrazione coinvolti nella migrazione delle cellule della cresta neurale in embrioni di Xenopus laevis

La famiglia del PDGF (fattore di crescita derivato dalle piastrine) è costituita da due recettori e quattro ligandi, in grado di formare omodimeri ed eterodimeri AB. Innumerevoli sono i ruoli svolti da tale fattore durante lo sviluppo e la vita adulta dei vertebrati, ma ancor più rilevante è la sua alterata espressione, comunemente riscontrata nei gliomi, un gruppo eterogeneo di tumori cerebrali altamente aggressivi e pressoché incurabili. Il progetto di ricerca, in cui s’inserisce il mio lavoro di tesi, nasce da una collaborazione con il Prof. Paolo Malatesta dell’Istituto Tumori di Genova; i suoi studi, condotti su modello murino, dimostrano che la sola sovraespressione del PDGF-B in progenitori neurali embrionali è in grado di indurre oligodendrogliomi, una specifica sottoclasse dei sopracitati tumori. Ulteriori esperimenti condotti in vivo ed in vitro suggeriscono il coinvolgimento del PDGF-B nella progressione tumorale, in particolare nella motilità e nell’invasività cellulare: le cellule di glioma indotto da PDGF-B mostrano, infatti, una straordinaria capacità d’infiltrare il parenchima cerebrale. Non sono ancora del tutto noti i meccanismi molecolari regolati dal PDGF-B, implicati nell’acquisizione della motilità e dell’invasività cellulare. Al fine di far luce su questi, ho utilizzato come modello di studio alternativo le cellule delle creste neurali (NCC) di embrioni di Xenopus laevis, data la loro affinità alle cellule cancerose in termini di transizione epitelio-mesenchimatica, invasività e migrazione a lunga distanza. Esperimenti preliminari di sovraespressione, mediante microiniezione di RNA messaggero di hPDGF-B su un solo lato dell’embrione, dimostrano che tale fattore è in grado di alterare il comportamento delle NCC, inducendone una migrazione ectopica ed eterocronica. Avendo ipotizzato un possibile coinvolgimento del PDGF-B nella regolazione dei segnali guida della migrazione delle NCC, ho sintetizzato sonde ad RNA antisenso per molecole, quali Cxcl-12 (Sdf-1), una chemochina, ed il suo recettore Cxcr-4, implicate nel processo di chemiotassi collettiva delle NCC, il fattore C3 del complemento ed il suo recettore C3aR, coinvolte nel fenomeno della co-attrazione, che mantiene coese le NCC durante la migrazione, ed infine le Neuropiline, necessarie a promuovere la segmentazione in “streams” migratori delle NCC. Queste molecole sono anche coinvolte nei processi di progressione tumorale dei gliomi. L’espressione di questi geni è stata quindi valutata su embrioni di Xenopus laevis microiniettati con mRNA di hPDGF-B. I risultati ottenuti suggeriscono un’alterazione dell’espressione di molecole coinvolte in processi di chemoattrazione ad opera della sovraespressione del PDGF-B. La migrazione alterata delle NCC potrebbe essere, inoltre, causata da cambiamenti nella loro motilità, forse dovuti ad un intervento diretto del PDGF-B sulle proprietà delle cellule stesse: al fine di valutare quest’ipotesi sto mettendo a punto saggi in vitro, utilizzando espianti di creste neurali sia sovraesprimenti hPDGF-B che “wild type”. Esperimenti di ibridazione in situ “whole mount” con sonde per marcatori di creste neurali mettono frequentemente in evidenza la migrazione di queste verso il presunto sito d’iniezione del PDGF-B. Per capire se tale fattore sia in grado, indipendentemente dall’influenza di altri, di attrarre le NCC, sto allestendo saggi di chemoattrazione in vivo ed in vitro. Ho inoltre caratterizzato il pattern d’espressione del Pdgf-b durante l’embriogenesi di Xenopus laevis, non ancora descritto in letteratura, per valutare un possibile ruolo fisiologico del fattore di crescita nel corso dello sviluppo. I risultati ottenuti, sommati agli studi su modello murino, potrebbero contribuire all’individuazione dei meccanismi molecolari alla base della tumorigenicità indotta da PDGF-B, con lo scopo di identificare nuove strategie terapeutiche

Danio rerio and Xenopus laevis embryos: alternative and valuable models to unveil new molecular mechanisms involved in glioma progression.

My project aimed at unveiling new molecular aspects involved in glioma progression. Since glutamate homeostasis deregulation seems to play a key role in glioblastoma (GBM) aggressiveness, the first part of the project was designed to deepen the role of the mitochondrial enzyme GLUD2 (glutamate dehydrogenase 2), which resulted differentially expressed between GBM patients with different recurrence-free survival after first surgery. I took advantage of Danio rerio embryos as an in vivo model to figure out GLUD2 possible function in regulating cell proliferation and survival, in order to evaluate its potential involvement in tumor growth. The second part of the project focused on another distinctive hallmark of gliomas, the infiltrative activity, which makes tumor complete surgical resection almost impossible. Given the urgent need of innovative therapeutic strategies, I tried to unveil new molecular mechanisms responsible of gliomas invasive nature. To this purpose, I investigated the role of PDGF-B in cell migration, since its up-regulation seemed to confer in vivo infiltrating properties on glioma tumor cells, exploiting Xenopus laevis neural crest cells (NCC) for their similarity to metastatic cancer cells. This model gave me the opportunity to achieve the final goal of my research, thus the possibility to discover new molecules involved in cell migration modulated downstream of PDGF-B signaling, paving the way towards the identification of new molecular therapeutic targets

Zebrafish Avatar to Develop Precision Breast Cancer Therapies

BACKGROUND: Zebrafish (Danio rerio) is a vertebrate that has become a popular alternative model for the cellular and molecular study of human tumors and for drug testing and validating approaches. Notably, zebrafish embryos, thanks to their accessibility, allow rapid collection of in vivo results prodromal to validation in the murine models in respect to the 3R principles. The generation of tumor xenograft in zebrafish embryos and larvae, or zebrafish avatar, represents a unique opportunity to study tumor growth, angiogenesis, cell invasion and metastatic dissemination, interaction between tumor and host in vivo avoiding immunogenic rejection, representing a promising platform for the translational research and personalized therapies.OBJECTIVE: In this mini-review we report recent advances in breast cancer research and drug testing that took advantage of the zebrafish xenograft model using both breast cancer cell lines and patient's biopsy.CONCLUSION: Patient derived xenograft, together with the gene editing, the omics biotechnology, the in vivo time lapse imaging and the high-throughput screening that are already set up and largely used in zebrafish, could represent a step forward towards precision and personalized medicine in the breast cancer research field