Relaxin ligand/receptor systems in the developing teleost fish brain : conserved features with mammals and a platform to address neuropeptide system functions

The relaxins (RLNs) are a group of peptide hormone/neuromodulators that can regulate a wide range of physiological processes ranging from reproduction to brain function. All the family members have originated from a RLN3-like ancestor via different rounds of whole genome and gene specific duplications during vertebrate evolution. In mammals, including human, the divergence of the different family members and the emergence of new members led to the acquisition of specific functions for the various relaxin family peptide and associated receptor genes. In particular, in mammals, it was shown, that the role of RLN3 is correlated to the modulation of arousal, stress responses, emotion, social recognition, and other brain functions, positioning this gene/peptide as a potential therapeutic target for neuropsychiatric disorders. This review highlights the evolutionary conservation of relaxin family peptide and receptor gene expression and their associated brain neural circuits. In the zebrafish, the expression pattern of the different relaxin family members has specific features that are conserved in higher species, including a likely similar functional role for the ancestral RLN3-like gene. The use of different model organisms, particularly the zebrafish, to explore the diversification and conservation of relaxin family ligands and receptor systems, provides a relatively high-throughput platform to identify their specific conserved or differential neuromodulatory roles in higher species including human

Apoptosis Induced by Piroxicam plus Cisplatin Combined Treatment Is Triggered by p21 in Mesothelioma

BACKGROUND: Malignant mesothelioma (MM) is a rare, highly aggressive tumor, associated to asbestos exposure. To date no chemotherapy regimen for MM has proven to be definitively curative, and new therapies for MM treatment need to be developed. We have previously shown in vivo that piroxicam/cisplatin combined treatment in MM, specifically acts on cell cycle regulation triggering apoptosis, with survival increase. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed, at molecular level, the apoptotic increase caused by piroxicam/cisplatin treatment in MM cell lines. By means of genome wide analyses, we analyzed transcriptional gene deregulation both after the single piroxicam or cisplatin and the combined treatment. Here we show that apoptotic increase following combined treatment is mediated by p21, since apoptotic increase in piroxicam/cisplatin combined treatment is abolished upon p21 silencing. CONCLUSIONS/SIGNIFICANCE: Piroxicam/cisplatin combined treatment determines an apoptosis increase in MM cells, which is dependent on the p21 expression. The results provided suggest that piroxicam/cisplatin combination might be tested in clinical settings in tumor specimens that express p21

Massive-Scale RNA-Seq Analysis of Non Ribosomal Transcriptome in Human Trisomy 21

Hybridization- and tag-based technologies have been successfully used in Down syndrome to identify genes involved in various aspects of the pathogenesis. However, these technologies suffer from several limits and drawbacks and, to date, information about rare, even though relevant, RNA species such as long and small non-coding RNAs, is completely missing. Indeed, none of published works has still described the whole transcriptional landscape of Down syndrome. Although the recent advances in high-throughput RNA sequencing have revealed the complexity of transcriptomes, most of them rely on polyA enrichment protocols, able to detect only a small fraction of total RNA content. On the opposite end, massive-scale RNA sequencing on rRNA-depleted samples allows the survey of the complete set of coding and non-coding RNA species, now emerging as novel contributors to pathogenic mechanisms. Hence, in this work we analysed for the first time the complete transcriptome of human trisomic endothelial progenitor cells to an unprecedented level of resolution and sensitivity by RNA-sequencing. Our analysis allowed us to detect differential expression of even low expressed genes crucial for the pathogenesis, to disclose novel regions of active transcription outside yet annotated loci, and to investigate a plethora of non-polyadenilated long as well as short non coding RNAs. Novel splice isoforms for a large subset of crucial genes, and novel extended untranslated regions for known genes—possibly novel miRNA targets or regulatory sites for gene transcription—were also identified in this study. Coupling the rRNA depletion of samples, followed by high-throughput RNA-sequencing, to the easy availability of these cells renders this approach very feasible for transcriptome studies, offering the possibility of investigating in-depth blood-related pathological features of Down syndrome, as well as other genetic disorders

Long non-coding RNA in neurons: New players in early response to BDNF stimulation

Brain-derived neurotrophic factor (BDNF) is a neurotrophin family member that is highly expressed and widely distributed in the brain. BDNF is critical for neural survival and plasticity both during development and in adulthood, and dysfunction in its signaling may contribute to a number of neurodegenerative disorders. Deep understanding of the BDNF-activated molecular cascade may thus help to find new biomarkers and therapeutic targets. One interesting direction is related to the early phase of BDNF-dependent gene expression regulation, which is responsible for the activation of selective gene programs that lead to stable functional and structural remodeling of neurons. Immediate-early coding genes activated by BDNF are under investigation, but the involvement of the non-coding RNAs is largely unexplored, especially the long non-coding RNAs (lncRNAs). lncRNAs are emerging as key regulators that can orchestrate different aspects of nervous system development, homeostasis, and plasticity, making them attractive candidate markers and therapeutic targets for brain diseases. We used microarray technology to identify differentially expressed lncRNAs in the immediate response phase of BDNF stimulation in a neuronal cell model. Our observations on the putative functional role of lncRNAs provide clues to their involvement as master regulators of gene expression cascade triggered by BDNF

STUDIO DELL’ESPRESSIONE GENICA DELLA PROTIMOSINA ALFA E DELLA RELASSINA-3 DURANTE LO SVILUPPO EMBRIONALE DEL TELEOSTEO Danio rerio

I meccanismi di regolazione della trascrizione genica e le funzioni biologiche della protimosina alfa e della relassina-3 sono studiati principalmente nei mammiferi. Alla protimosina alfa sono state accreditate diverse funzioni tra cui un ruolo attivo nella proliferazione e sopravvivenza cellulare, mentre la relassina-3, funziona come neurotrasmettitore per un gruppo ristretto di neuroni detto nucleo incerto. Con l'obiettivo di allargare la conoscenza sul profilo d'espressione genica della protimosina alfa e della relassina-3 in altre classi di vertebrati, e in particolare durante lo sviluppo embrionale, abbiamo usato come modello sperimentale il pesce teleosteo Danio rerio ottenendo i seguenti risultati: 1. Protimosina alfa: abbiamo dimostrato che il gene per la protimosina alfa risulta duplicato in Danio rerio e che entrambi i geni identificati si esprimono durante lo sviluppo embrionale. A partire dalla duplicazione, i due geni hanno diversificato i meccanismi di regolazione trascrizionale poiché i profili d'espressione sono simili ma non identici. Nel complesso, i due geni mostrano che l'espressione non è ubiquitaria, ma riguarda territori specifici. L'espressione osservata in alcuni territori, come l'abbozzo della coda, fa ipotizzare che la protimosina alfa sia coinvolta nei meccanismi di proliferazione cellulare, mentre in altri, come le creste neurali, è presumibilmente coinvolta nella sopravvivenza cellulare, mostrando che tali funzioni sono conservate nei vertebrati. 2. Relassina-3: anche in questo caso abbiamo dimostrato una duplicazione genica che ha portato all'esistenza di due paraloghi, rln3a e rln3b. Studiandone il profilo d'espressione abbiamo raggiunto importanti risultati nella neuroanatomia dei pesci. Infatti entrambi i geni, nelle prime fasi dello sviluppo del cervello, sono espressi in tutto il territorio neurale, mentre nelle fasi finali del periodo di faringula restringono drasticamente la loro espressione a piccoli gruppi di cellule nella regione mesencefalica. Analisi di sezioni trasversali unite all'uso di opportuni marcatori territoriali ci hanno fatto concludere che si tratta di cellule del grigio periacqueduttale, una regione del cervello fino ad ora mai descritta nel pesce zebra. Mentre rln3b si esprime esclusivamente in questo territorio anche agli stadi successivi, rln3a, a partire dallo stadio larvale, si esprime anche in un altro gruppo di cellule situato nella regione rombencefalica intorno al quarto ventricolo. La relazione neuroanatomica di questo gruppo di cellule con il rafe dorsale e superiore ed il locus coeruleus dimostra che appartiene alla regione dei ponti. Queste osservazioni ci hanno fatto ipotizzare che si tratti di una struttura omologa al nucleo incerto dei mammiferi. L’ipotesi è stata confermata mediante l'analisi dei territori d'espressione del gene crhr1, che nel cervello di ratto caratterizza proprio il nucleo incerto. Tali evidenze sperimentali sono le prime riguardanti l'esistenza del nucleo incerto nel cervello dei pesci. L'espressione del gene rln3a nel pesce zebra dimostra che la sua funzione come neurotrasmettitore è così importante da mantenersi perfettamente conservata nell'evoluzione dei vertebrati. Inoltre, dati preliminari sull'espressione genica dei recettori della relassina-3 mostrano che i circuiti neurali legati alla risposta allo stress, di cui fa parte il nucleo incerto, sono potenzialmente funzionali già nelle prime fasi di vita dell'organismo

Role of the transcription factor EGR1 in the neuronal survival

Cells respond to neural stimuli through the rapid and transient induction of a set of genes called immediate early genes (IEGs). Among these genes, EGR1 (early growth response gene 1) encodes for a transcription factor known to be involved in different biological functions such as cell growth, differentiation and apoptosis. In the neuronal development, EGR1 has been hypothesized to have a role in differentiation, neuronal plasticity, and in the development of learning and memory. The scope of our study is to evaluate the involvement of EGR1 in the neuronal cell proliferation, survival and death. To this aim, we used SH-SY5Y neuroblastoma cell line and CRISPR-Cas9 technology to generate an EGR1 knock-out (KO) neuronal cell model. EGR1-KO cells show clear morphological differences, and higher proliferation and migration rate compared to WT cells. Under different stress conditions, such as serum withdrawal and H2O2-induced oxidative stress, KO cells have higher survival rate than WT cells. A preliminary molecular analysis demonstrated that KO cells react to stress-induced conditions by triggering autophagy process in contrast to WT cells. Therefore, EGR1 knockout cells may represent an interesting model for studying the molecular mechanisms regulated by EGR1 for the proper development, differentiation and survival of neurons

Role of the transcription factor EGR1 in neuronal differentiation

Background: immediate early genes encode transcription factors which combine early signaling events with long-term changes in gene expression. They are involved in many cellular processes, including differentiation and proliferation. The transcription factor EGR1 has been hypothesized to have a role in many biological processes such as cell growth, differentiation and apoptosis during neuronal differentiation. Genes regulated by EGR1 are also involved in neuronal plasticity and in the development of learning and memory. The expression of EGR1 is often altered in neurodegenerative diseases and psychiatric disorders. However, its mechanism of action is not fully understood. Method: We used SH-SY5Y cell line as a neuronal cell model, since SH-SY5Y cells can be differentiated to a more mature neuron-like phenotype using retinoic acid (RA). We analyzed the expression levels of EGR1 after RA treatment using qPCR and Western Blot analysis. Then, in order to characterize the role of EGR1 in neuronal differentiation we used CRISPR-Cas9 technology to generate a knockout cell (KO) line for EGR1 gene. Results: EGR1 mRNA and protein levels increase significantly during the first two days of RA treatment, leading to the idea of an early involvement of EGR1 in RA-induced neuronal differentiation. Interestingly, EGR1 KO cells undergo to cell death after RA treatment. Molecular analysis of the EGR1 KO model showed a dysregulation of many differentiation markers, including those related to synaptogenesis. Conclusions: Our findings suggest a key role of EGR1 in the proper development and differentiation of neurons, in particular, in the relationship between neuronal differentiation and survival