Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: Effects of CREB pathway inhibition

JRC.I.5-Systems Toxicolog

Approccio proteomico integrato allo studio dei domini PDZ

La proteomica è una nuova disciplina, nata dall’esigenza di studiare in modo sistematico tutte le proteine prodotte nei vari organismi, di cui è in corso oppure è già stato completato il sequenziamento del genoma. In passato, allo scopo di visualizzare un’espressione proteica differenziale in condizioni o stati biologici diversi venivano utilizzati essenzialmente metodi di elettroforesi su gel bidimensionale. Con l’emergere di nuove potenti tecnologie analitiche, come la spettrometria di massa, e con lo sviluppo di metodi bioinformatici per analizzare le sequenze genomiche e dedurre la composizione amminoacidica dei prodotti genici, la proteomica ha esteso il suo campo d’azione all’analisi della funzione dei prodotti genici (“genomica funzionale”), includendo studi d’identificazione, di localizzazione, di espressione e d’interazione tra le proteine. In particolare, la creazione di una mappa d’interazioni proteina-proteina di una cellula potrà essere di gran valore per la comprensione della biologia della cellula stessa. Il riconoscimento e il legame tra le proteine è alla base della formazione di complessi multiproteici, fondamentali per molte attività della cellula come lo sviluppo, la trasduzione del segnale, l’adesione e la comunicazione con altre cellule. Un gran numero di interazioni sono mediate da famiglie di “domini” di legame proteici, che sono porzioni di proteine, ripiegate indipendentemente e autonomamente dal resto della molecola, comprendenti circa 40-200 aminoacidi. Sono domini non catalitici che legano specificamente e reversibilmente una regione più o meno estesa della molecola bersaglio, tramite una cavità specializzata per il riconoscimento. Per questo lavoro di tesi, ho concentrato il mio studio su una particolare famiglia di domini di legame, che sono molto frequenti nelle proteine e partecipano alla formazione di svariati complessi multiproteici. I domini proteici “PDZ” sono stati descritti in origine come elementi strutturali conservati in tre proteine, PSD95 (componente di un complesso sotto la membrana post-sinaptica), DLG (un soppressore tumorale) e ZO-1 (presente nelle giunzioni strette), e successivamente sono stati individuati in molte proteine dei metazoi, soprattutto a livello delle membrane cellulari, dove svolgono un ruolo di “adattatori” o di “impalcature” alle quali le altre proteine sono associate. Allo scopo di caratterizzare le modalità con cui i domini PDZ riconoscono e legano i loro bersagli, ho isolato le regioni codificanti per alcuni domini PDZ presenti in proteine di differenti organismi: uomo, topo e zebrafish. Ho utilizzato quindi un approccio proteomico integrato per studiare: 1) la specificità di riconoscimento dei vari domini, 2) le proteine bersaglio con cui possono interagire, 3) i componenti degli eventuali complessi multiproteici, di cui fanno parte. Ho quindi analizzato repertori di peptidi a sequenze casuali, esposti su capside fagico (“peptide phage-display”) oppure librerie fagiche di prodotti di cDNA (cDNA-phage display), identificando i peptidi che vengono legati preferenzialmente da ciascun PDZ. Inoltre partendo da estratti proteici ottenuti da cervello di topo, ho selezionato mediante cromatografia di affinità le proteine che rimangono associate ai PDZ in vitro e ho provato a confermare in vivo queste interazioni. Alcune delle proteine bersaglio sono state identificate mediante spettrometria di massa, nell’ambito di un progetto di collaborazione. L’uso di approcci differenti ha permesso di capire i vantaggi e i limiti delle varie tecnologie, mettendo in evidenza la necessità di comparare e integrare i vari risultati, allo scopo di avere un quadro completo di come questi domini modulano le attività delle proteine all’interno delle cellule

Dissecting the interaction network of a multi-PDZ protein

Multimodular scaffolds, containing multiple copies of binding domains, such as multi-PDZ proteins, play a crucial role in the formation and localization of complex multicomponent assemblies, which are fundamental for many activities of the cell. Unravelling the components of the networks, mediated by such multivalent binding proteins, is essential for understanding how signal transduction and intracellular pathways are organized. During my doctorate research, I analyzed the network mediated by the protein CIPP, “channel-interacting PDZ protein”, a typical example of multi-PDZ protein, engaged in the interaction with several channels and receptors, at membrane level. CIPP belongs to a large family of proteins, composed of several isoforms, whose tissue specific expression I have analyzed by RT-PCR. In particular, I concentrated my study on an isoform containing four PDZ domains and I analyzed its brain-specific expression, using in situ hybridization assays. In order to identify the precise components of the protein interaction complex mediated by CIPP PDZ domains, I characterized two novel cytoplasmic CIPP interactors: the proteins Cypin and IRSp53, both widely involved in mechanisms of cytoskeletal remodelling. Their direct and specific interaction with CIPP-PDZ1 and CIPP-PDZ2, respectively, was verified using multiple biochemical binding assays. The binding occurrence was also proved with the full length CIPP recombinant protein and, by generating mutant constructs, I showed the importance of an intact canonical PDZ binding motif at the carboxyl termini of the ligands. Immunolocalization analysis on cultured cells confirmed the reciprocal interactions and, moreover, showed that IRSp53, following PDZ-mediated interaction with CIPP, was able to induce the formation of punctate cytoplasmic clusters. These CIPP-containing puncta were not associated with endocytic vesicles, but they were simple protein assemblies, containing CIPP, IRSp53 and Cypin. Finally, I established that IRSp53 and Cypin were able to directly interact one with each other, through specific regions, independently from the PDZ-mediated binding to CIPP. Interestingly, their clustering in cytoplasmic puncta occurred only when also CIPP was present. All the information derived from these studies suggest that CIPP-PDZ domains are responsible for the formation of a tripartite CIPP-IRSp53-Cypin complex, which could contact membrane channels or other receptors, such as NMDA receptors, and mediate signal transduction inside the cell to the final cytoskeletal targets

Channel-interacting PDZ protein "CIPP" interacts with proteins involved in cytoskeletal dynamics

Neuronal CIPP (channel-interacting PDZ protein) is a multivalent PDZ protein that interacts with specific channels and receptors highly expressed in the brain. It is composed of four PDZ domains that behave as a scaffold to clusterize functionally connected proteins. In the present study, we selected a set of potential CIPP interactors that are involved directly or indirectly in mechanisms of cytoskeletal remodelling and membrane protrusion formation. For some of these, we first proved the direct binding to specific CIPP PDZ domains considered as autonomous elements, and then confirmed the interaction with the whole protein. In particular, the small G-protein effector IRSp53 (insulin receptor tyrosine kinase substrate protein p53) specifically interacts with the second PDZ domain of CIPP and, when co-transfected in cultured mammalian cells with a tagged full-length CIPP, it induces a marked reorganization of CIPP cytoplasmic localization. Large punctate structures are generated as a consequence of CIPP binding to the IRSp53 C-terminus. Analysis of the puncta nature, using various endocytic markers, revealed that they are not related to cytoplasmic vesicles, but rather represent multi-protein assemblies, where CIPP can tether other potential interactors

www.mdpi.com/journal/ijms Role of Btg2 in the Progression of a PDGF-Induced Oligodendroglioma Model

Abstract: Tumor progression is a key aspect in oncology. Not even the overexpression of a powerful oncogenic stimulus such as platelet derived growth factor-B (PDGF-B) is sufficient per se to confer full malignancy to cells. In previous studies we showed that neural progenitors overexpressing PDGF-B need to undergo progression to acquire the capability to give rise to secondary tumor following transplant. By comparing the expression profile of PDGF-expressing cells before and after progression, we found that progressed tumors consistently downregulate the expression of the antiproliferative gene Btg2. We therefore tested whether the downregulation of Btg2 is sufficient and necessary for glioma progression with loss and gain of function experiments. Our results show that downregulation of Btg2 is not sufficient but is necessary for tumor progression since the re-introduction of Btg2 in fully progressed tumors dramatically impairs their gliomagenic potential. These results suggest an important role of Btg2 in glioma progression. Accordingly with this view, the analysis of public datasets of human gliomas showed that reduced level of Btg2 expression correlates with a significantly worse prognosis

Channel-interacting PDZ protein “CIPP” interacts with proteins involved in cytoskeletal dynamics

International audienceNeuronal CIPP is a multivalent PDZ protein that interacts with specific channels and receptors, highly expressed in the brain. It is composed of four PDZ domains that behave as a scaffold to clusterize functionally connected proteins. In this study, we selected a set of potential CIPP interactors that are directly or indirectly involved in mechanisms of cytoskeletal remodeling and membrane protrusions formation. For some of these, we first proved the direct binding to specific CIPP PDZ domains considered as autonomous elements, and then confirmed the interaction with the whole protein. In particular, the small G-protein effector IRSp53 (insulin receptor tyrosine kinase substrate protein p53) specifically interacts with the second PDZ domain of CIPP and, when co-transfected in mammalian cultured cells with a tagged full-length CIPP, it induces a marked reorganization of CIPP cytoplasmic localization. Large punctate structures are generated as a consequence of CIPP binding to IRSp53 carboxy-terminus. Analysis of the puncta nature, using various endocytic markers, revealed that they are not related to cytoplasmic vesicles, rather represent multi-protein assemblies, where CIPP can tether other potential interactors

Assessment of acute and chronic toxicity of doxorubicin in human induced pluripotent stem cell-derived cardiomyocytes

The present study assesses acute and chronic toxicity of doxorubicin in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), with the aim to obtain in vitro biomarkers that can be used as readouts to predict in vivo cardiotoxicity. Possible acute toxicity was investigated by assessing effects on the beating rate and the field potential duration (FPD) of doxorubicin-exposed cardiomyocytes by measuring electrical activity using multi-electrode array (MEA) analyses. No effects on the beating rate and FPD were found at concentrations up to 6 mu M, whereas at 12 mu M no electrical activity was recorded, indicating that the cardiomyocytes stopped beating. Acute and chronic effects of doxorubicin on mitochondria, which have been reported to be affected in doxorubicin-induced cardiotoxicity, were assessed using high content imaging techniques. To this end hiPSC-CMs were exposed to 150 or 300 nM doxorubicin using both single dosing (3 h and 2 days) and repetitive dosing (3 times, of 2 days each), including washout studies to assess delayed effects (assessment at day 14) and effects on cell number, mitochondrial density, mitochondrial membrane potential, mitochondrial superoxide levels and mitochondrial calcium levels were assessed. No effects of doxorubicin were found on mitochondrial density and mitochondrial superoxide levels, whereas doxorubicin reduced cell survival and slightly altered mitochondrial membrane potential and mitochondrial calcium levels, which was most profound in the washout studies. Altogether, the results of the present study show that concentrations of doxorubicin in the micromolar range were required to affect electrical activity of hiPSC-CMs, whereas nanomolar concentrations already affected cell viability and caused mitochondrial disturbances. Integration of these data with other in vitro data may enable the selection of a series of in vitro biomarkers that can be used as readouts to screen chemicals for possible cardiotoxicit

Assessment of acute and chronic toxicity of doxorubicin in human induced pluripotent stem cell-derived cardiomyocytes

The present study assesses acute and chronic toxicity of doxorubicin in human induced pluripotent stem cellderived cardiomyocytes (hiPSC-CMs), with the aim to obtain in vitro biomarkers that can be used as readouts to predict in vivo cardiotoxicity. Possible acute toxicity was investigated by assessing effects on the beating rate and the field potential duration (FPD) of doxorubicin-exposed cardiomyocytes by measuring electrical activity using multi-electrode array (MEA) analyses. No effects on the beating rate and FPD were found at concentrations up to 6 μM, whereas at 12 μM no electrical activity was recorded, indicating that the cardiomyocytes stopped beating. Acute and chronic effects of doxorubicin on mitochondria, which have been reported to be affected in doxorubicin-induced cardiotoxicity, were assessed using high content imaging techniques. To this end hiPSCCMs were exposed to 150 or 300 nM doxorubicin using both single dosing (3 h and 2 days) and repetitive dosing (3 times, of 2 days each), including washout studies to assess delayed effects (assessment at day 14) and effects on cell number, mitochondrial density, mitochondrial membrane potential, mitochondrial superoxide levels and mitochondrial calcium levels were assessed. No effects of doxorubicin were found on mitochondrial density and mitochondrial superoxide levels, whereas doxorubicin reduced cell survival and slightly altered mitochondrial membrane potential and mitochondrial calcium levels, which was most profound in the washout studies. Altogether, the results of the present study show that concentrations of doxorubicin in the micromolar range were required to affect electrical activity of hiPSC-CMs, whereas nanomolar concentrations already affected cell viability and caused mitochondrial disturbances. Integration of these data with other in vitro data may enable the selection of a series of in vitro biomarkers that can be used as readouts to screen chemicals for possible cardiotoxicity.JRC.F.3-Chemicals Safety and Alternative Method

Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: effects of CREB pathway inhibition

According to the new paradigm shift in toxicity testing, acquisition of knowledge on the mechanisms underlying the toxicity of chemicals, such as perturbations of biological pathways, is of primary interest. Moreover, pluripotent stem cells (PSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), offer a unique opportunity to derive physiologically relevant human cell types to be used in these mechanistic studies. In the present study, we compared the neuronal differentiation propensity of hESCs and hiPSCs aiming to develop tolls for mechanistic neurotoxicity testing. The gene expression and signaling pathway analyses demonstrate the activation of a similar neuronal signature in the two cellular models, particularly indicating that the neuronal survival related cAMP responsive element binding protein (CREB) pathway gets activated upon differentiation. Furthermore, analysis of CREB pathway inhibition, using 2-naphthol-AS-E-phosphate, shows a decrease in neuronal cells as well as an inhibition of neurite outgrowth, synaptogenesis and impairment of electrical activity. These data indicate that inhibition of the CREB pathway can be related to relevant endpoints for neurotoxicity testing in our in vitro cell model, and, as such, qualify the use of this cellular model for mechanism-based toxicity testingJRC.I.5-Systems Toxicolog