Regolazione del recettore GPR17 in cellule precursori degli oligodendrociti

La Sclerosi multipla (SM) è una patologia infiammatoria cronica del sistema nervoso centrale (SNC), caratterizzata da una progressiva perdita della guaina mielinica, la quale garantisce un’adeguata trasmissione dell’impulso nervoso tramite il rivestimento degli assoni . La patologia risulta caratterizzata dalla presenza, a livello del SNC, di placche “sclerotiche”, come conseguenza del rilascio di molecole pro-infiammatorie che vanno ad attaccare la guaina mielinica, così come le cellule deputate alla loro produzione, gli oligodendrociti. Quest’ultime sono cellule mature derivanti da precursori gliali indifferenziati, i precursori degli oligodendrociti (OPC), che si ritrovano a livello del SNC umano dalla nascita. Questi precursori vanno incontro ad un preciso programma di proliferazione, migrazione, differenziazione e mielinizzazione, per produrre infine la guaina isolante degli assoni. Lo sviluppo degli oligodendrociti in vitro progredisce attraverso stadi distinti, caratterizzati da una combinazione molto dinamica di fattori di trascrizione e specifici fenotipi antigenici. Tra i numerosi fattori implicati nel processo di mielinizzazione ritroviamo il GPR17. Il GPR17 è un recettore accoppiato a proteine G (GPCR), prevalentemente di tipo inibitorio; si tratta di un recettore dualistico, che risponde a due famiglie distinte di ligandi: i nucleotidi uridinici (UDP, UDP-glucosio), e cisteinil-leucotrieni (LTD4, LTC4, LTE4), con affinità rispettivamente nell’ordine del micromolare e del nanomolare. Durante il differenziamento degli OPC, l’espressione del GPR17 segue una finestra spazio-temporale molto precisa: è ristretta agli stadi iniziali di maturazione mentre risulta nulla negli oligodendrociti maturi. È stato, inoltre, evidenziato che una forzata espressione del recettore negli OPC comporta un rallentamento della maturazione, e di conseguenza un blocco della produzione di mielina, portando a morte degli oligodendrociti. Il coinvolgimento del GPR17 nella SM viene confermato dalla presenza di un up-regulation recettoriale a livello delle placche sclerotiche. Tenendo conto di questi dati, è stato ipotizzato che, al fine di una completa maturazione degli OPC, sia necessario uno spegnimento del recettore GPR17, attraverso un opportuno meccanismo di desensitizzazione, internalizzazione e down–regulation. La desensitizzazione di un GPCR, ovvero la perdita di funzionalità recettoriale, in seguito alla prolungata esposizione ad un agonista, prevede di norma, come primo step, una fosforilazione del recettore su residui di serina e treonina, da parte di chinasi specifiche, denominate GRK, dopo associazione delle stesse al recettore; questo determina un aumento di affinità del recettore verso la proteina citoplasmatica β-arrestina, la quale ne promuove il distacco dalla proteina G e la sua internalizzazione in endosomi. Questo lavoro di tesi si prefigge di indagare i meccanismi di regolazione del recettore GPR17 durante la maturazione degli OPC, ed in particolare il ruolo di specifiche GRK in tale processo. Tra i vari sottotipi delle GRK (classificate da 1 a 6), il focus si è rivolto verso la GRK2 e la GRK5. I livelli di GRK2 sono stati, infatti, ritrovati significativamente ridotti proprio in pazienti affetti da SM, mentre la GRK5 risulta funzionalmente alterata in patologie infiammatorie croniche. Da qui l’ipotesi di un loro possibile coinvolgimento nella regolazione funzionale del GPR17 negli OPC. Gli OPC sono stati isolati da colture gliali della corteccia di ratto, quindi differenziati e raccolti a diversi stadi di differenziamento. Attraverso analisi Real Time PCR è stato confermato l’andamento di espressione del trascritto del GPR17 durante il differenziamento; dagli stessi esperimenti è inoltre risultato che le GRK 2 e 5 presentano un livello basale (negli OPC non differenziati) più alto del GPR17, ma la loro espressione ha un andamento molto simile a quello del recettore, raggiungendo un picco nei pre-oligodendrociti, per poi diminuire negli oligodendrociti maturi. Abbiamo poi verificato, se, in seguito a stimolazione del GPR17 con i suoi agonisti, ci fosse fosforilazione del recettore. I risultati ottenuti attraverso un saggio di co-immunoprecipitazione e immunoblotting hanno evidenziato che, dopo trattamento dei pre-oligodendrociti per 5 min con UDP-glucosio o con LTD4, il GPR17 risulta fosforilato su residui di treonina, e , in misura minore, su quelli di serina. Per indagare il coinvolgimento delle singole GRK nel processo di fosforilazione, abbiamo valutato l’associazione tra il recettore e le due chinasi. Da saggi di co-immunoprecipitazione è risultato che la GRK5 si associa fisicamente con il GPR17 preferenzialmente quando questo è stimolato da UDP-glucosio; al contrario in seguito alla stimolazione con LTD4, l’associazione del recettore avviene con entrambe le GRK, anche se in misura preferenziale con la GRK2. La funzionalità del GPR17 è stata poi indagata valutando le sue cinetiche di desensitizzazione, mediante saggi funzionali di cAMP. Gli OPC sono stati pre-trattati con alte concentrazioni di UDP-glucosio e LTD4 per vari tempi, lavati, e stimolati nuovamente con gli agonisti in presenza di forskolina. Il saggio è stato effettuato sia al picco massimo di espressione del GPR17, sia allo stadio nel quale l’espressione del recettore tende a diminuire. Nel primo caso, si è osservato che entrambi gli agonisti, leucotrienici e nucleotidici, promuovono una desensitizzazione omologa del recettore con andamento tempo-dipendente. La cinetica di desensitizzazione appare più veloce quando indotta dall’agonista leucotrienico. Nei pre-oligodendrociti maturi, invece, il fenomeno di desensitizzazione è risultato appena significativo solo dopo 2 ore di pre-trattamento con l’agonista uridinico o leucotreinico. Questi risultati sono in linea con una diminuizione d’espressione delle GRK in questo stadio di maturazione degli OPC. Infine, abbiamo verificato il ruolo della GRK2 nel meccanismo di desensitizzazione del recettore mediante l’uso di un’inibitore specifico per tale chinasi. L’utilizzo dell’inibitore GRK2 ha bloccato la desensitizzazione del GPR17 indotta dall’agonista leucotrienico, mentre non ha mostrato effetto in seguito a stimolazione con l’agonista uridinico. Questi dati dimostrano che, analogamente a quanto dimostrato in precedenza in cellule transfettate, anche in cellule primarie la GRK2 ha un ruolo cruciale nella desensitizzazione del sito leucotrienico del GPR17. Al momento sono in corso studi per valutare la GRK implicata nella regolazione del sito purinergico. In conclusione, i risultati ottenuti dimostrano che negli OPC il recettore GPR17, in seguito a stimolo con UDP-glucosio o LTD4, va incontro a fosforilazione, su residui di serina e treonina, e a desensitizzazione tempo-dipendente. Tale fenomeno sembra coinvolgere principalmente la GRK2 per quanto riguarda il sito leucotrienico: LTD4 induce, infatti, l’associazione preferenziale del recettore con la tale chinasi, e media desensitizzazione solo in presenza di GRK2 funzionalmente attiva. Dati preliminari suggeriscono invece che il ligando purinergico induca desensitizzazione recettoriale attraverso la GRK5. Tali risultati dimostrano che lo stesso recettore, attivato da classi di ligandi chimicamente diversi tra loro, può seguire vie intracellulari diverse. I dati risultano utili nella comprensione della regolazione del GPR17 durante la mielinizzazione degli OPC

TUMOR NECROSIS FACTOR ALPHA TRIGGERS OSTEOGENESIS THROUGH THE INVOVLVEMENT OF Gs-COUPLED RECEPTOR SIGNALS

Tumor Necrosis Factor alpha (TNF-α) plays a role in several chronic immune and inflammatory diseases, where inhibition of TNF has led to significant clinical improvement. Actually, this cytokine is involved in bone healing by affecting mesenchymal stem cell (MSC) behaviour in a dose- and time-dependent manner1,2. Indeed, in the early inflammatory phase after fracture, low doses of TNF-α are required to favour MSC migration, survival and differentiation, thus initiating bone repair. At high dose, in the chronic uncontrolled phase of inflammation, the same cytokine has destructive effects on bone and contribute to bone loss1,2. As other soluble factors released in cell microenvironment, the cytokine modulates expression and functioning of different G protein coupled receptors (GPCRs) and of their regulatory proteins (GPCR regulated kinases, GRKs)3, thus dictating the final biological outcome of these receptor proteins in controlling bone anabolic processes. Herein, we investigated the effects of TNF-α low doses on the expression and functional responsiveness of A2B adenosine receptor (A2B AR), a Gs-coupled puringergic receptor that controls mesenchymal stem cell (MSC) differentiation to osteoblasts4,5. In our hands, TNF-α exerted a pro-differentiating action on MSCs, pushing towards an osteoblast phenotype, and without any effects on cell proliferation. The cytokine increased the A2B AR-mediated pro-osteogenic effects, through the A2B AR desensitization impairment mediated by GRK2 inhibition. These data i) support the anabolic effect of sub-massimal concentration of TNF-α in bone reparative processes and ii) demonstrate that the cytokine regulates GPCR responses by interfering with desensitization machinery and potentiating in turn the anabolic responses evoked by Gs-GPCRs. Overall these results indicated that manipulating MSC local environment by lregulates membrane receptors favouring bone remodelling

Historical Single-Use Heritage Assets: A Treasure to be Catalogued and Preserved

Special-purpose historical assets are those built in the twentieth century, in certain environmental, social, industrial, economic, infrastructural contexts, which carried out a single function. This unique function determined the dimensions, partitions, accesses and, in short, the very particular and specific characteristics that excluded their reuse. Having lost their usefulness, they are destined in the space of a few years to disap­pear, to be demolished, their past lost without leaving any memory or mark, because they are not protected by current legislation.The proposed cataloging and conservation of these structures is aimed at focus­ing attention on the afore-mentioned historical assets and protecting these significant testimonies not only from an architectural perspective but also from that of the history of work, popular culture and social aggregation.Gli edifici finalizzati sono quelle costruzione sorte, nel Novecento, in determinati contesti ambientali, sociali, industriali, economici, infrastrutturali, che svolgevano una unica funzione. Questa unica funzione ne ha determinato le dimensioni, le ripartizioni, gli accessi e, in sintesi, le caratteristiche così particolari e specifiche che fanno esclu­dere riutilizzi. Perdendo la loro utilità, sono destinati nel giro di pochi anni a scomparire, ad essere demoliti, a perdersi nella memoria senza lasciare spoglie anche perché non oggetto di tutela da parte delle Normative vigenti.La proposta di catalogazione e conservazione degli edifici finalizzati è finalizzata a porre l’attenzione sui suddetti manufatti e a tutelare queste testimonianze significative dal punto di vista non solo architettonico ma anche della storia del lavoro, della cultura popolare, della aggregazione sociale

Evidence of a low-temperature dynamical transition in concentrated microgels

A low-temperature dynamical transition has been reported in several proteins. We provide the first observation of a `protein-like' dynamical transition in nonbiological aqueous environments. To this aim we exploit the popular colloidal system of poly-N-isopropylacrylamide (PNIPAM) microgels, extending their investigation to unprecedentedly high concentrations. Owing to the heterogeneous architecture of the microgels, water crystallization is avoided in concentrated samples, allowing us to monitor atomic dynamics at low temperatures. By elastic incoherent neutron scattering and molecular dynamics simulations, we find that a dynamical transition occurs at a temperature $T_d\sim250$~K, independently from PNIPAM mass fraction. However, the transition is smeared out on approaching dry conditions. The quantitative agreement between experiments and simulations provides evidence that the transition occurs simultaneously for PNIPAM and water dynamics. The similarity of these results with hydrated protein powders suggests that the dynamical transition is a generic feature in complex macromolecular systems, independently from their biological function

Carnosol controls the human glioblastoma stemness features through the epithelial-mesenchymal transition modulation and the induction of cancer stem cell apoptosis

A high cell proliferation rate, invasiveness and resistance to chemotherapy are the main features of glioblastoma (GBM). GBM aggressiveness has been widely associated both with a minor population of cells presenting stem-like properties (cancer stem-like cells, CSCs) and with the ability of tumor cells to acquire a mesenchymal phenotype (epithelial-mesenchymal transition, EMT). Carnosol (CAR), a natural inhibitor of MDM2/p53 complex, has been attracted attention for its anti-cancer effects on several tumor types, including GBM. Herein, the effects of CAR on U87MG-derived CSC viability and stemness features were evaluated. CAR decreased the rate of CSC formation and promoted the CSC apoptotic cell death through p53 functional reactivation. Moreover, CAR was able to control the TNF-α/TGF-β-induced EMT, counteracting the effects of the cytokine on EMT master regulator genes (Slug, Snail, Twist and ZEB1) and modulating the activation of miR-200c, a key player in the EMT process. Finally, CAR was able to increase the temozolomide (TMZ) anti-proliferative effects. These findings demonstrate that CAR affected the different intracellular mechanism of the complex machinery that regulates GBM stemness. For the first time, the diterpene was highlighted as a promising lead for the development of agents able to decrease the stemness features, thus controlling GBM aggressiveness

New insights into the anticancer activity of carnosol: P53 reactivation in the U87MG human glioblastoma cell line

Glioblastoma multiforme (GBM) is an aggressive brain tumour with high resistance to radio- A nd chemotherapy. As such, increasing attention has focused on developing new therapeutic strategies to improve treatment responses. Recently, attention has been shifted to natural compounds that are able to halt tumour development. Among them, carnosol (CAR), a phenolic diterpene present in rosemary, has become a promising molecule that is able to prevent certain types of solid cancer. However, no data are available on the effects of CAR in GBM. Here, CAR activity decreased the proliferation of different human glioblastoma cell lines, particularly cells that express wild type p53. The p53 pathway is involved in the control of apoptosis and is often impaired in GBM. Notably, CAR, through the dissociation of p53 from its endogenous inhibitor MDM2, was able to increase the intracellular p53 levels in GBM cells. Accordingly, functional reactivation of p53 was demonstrated by the stimulation of p53 target genes' transcription, the induction of apoptosis and cell cycle blockade. Most importantly, CAR produced synergistic effects with temozolomide (TMZ) and reduced the restoration of the tumour cells' proliferation after drug removal. Thus, for the first time, these data highlighted the potential use of the diterpene in the sensitization of GBM cells to chemotherapy through a direct re-activation of p53 pathway. Furthermore, progress has been made in delineating the biochemical mechanisms underlying the pro-apoptotic effects of this molecule

G PROTEIN-COUPLED RECEPTOR DESENSITISATION REGULATES STEM CELL DIFFERENTIATION

G-protein coupled receptors (GPCRs) play a key role in many complex biological processes, including regulation of stem cell pluripotency and differentiation. Signal transduction pathways that are activated during stem cell renewal and differentiation are shared, cross-activated or synergistic with GPCR stimulation [1]. Regulation of GPCR responses involved the activation of desensitization machinery, which started with phosphorylation of agonist-activated receptor by second messenger-dependent and/or GPCR kinases (GRKs)[1]. Besides controlling receptor responsiveness, GRKs can also act as agonist-regulated scaffolds assembling macromolecular signalosomes in the receptor environment, thereby contributing to signal propagation from cytosol to nucleus, and controlling gene transcription machinery [2]. Recent evidence suggests that the desensitization machinery fulfils a vital role in regulating cellular responses to GPCRs, and that changes in expression/functioning of these regulatory proteins may be crucial in the control of cell differentiation program [3]. These data are consistent with the notion that GPCR responsiveness may be differentially regulated during cell differentiation. In our hands, two different cellular models (oligodendrocyte precursor cells, OPCs, and mesenchymal stem cells, MSCs) were used to investigate the role of the GPCR desensitisation machinery in stem cell differentiation. During OPC differentiation, defective control of the membrane receptor GPR17 has been suggested to block cell maturation and impairs remyelination under demyelinating conditions [4]. Here we show, for the first time, a role for Murine double minute 2 (Mdm2), a ligase previously involved in ubiquitination/degradation of p53 protein. In maturing OPCs, the inhibition of Mdm2-p53 interactions increased GRK2 sequestration by Mdm2, leading to impaired GPR17 down-regulation and OPC maturation block. In MSCs, the A2B adenosine receptor (A2BAR) has been recently emerged as the major AR involved in osteoblastogenesis [5]. Proinflammatory cytokines, such as Tumour Necrosis Factor- (TNF-, have been demonstrated to regulate MSC differentiation and bone remodelling. Herein, we show that TNF- diminished GRK2 levels in MSCs, thus blocking A2BAR desensitization. As a result, TNF- enhanced the A2BAR-mediated responses and favoured MSC differentiation to osteoblasts in response to receptor agonists. The findings get new insights for discovering of the signals at the basis of cell differentiation

Spatio-temporal patterns of genetic diversity in the Mediterranean striped dolphin (Stenella coeruleoalba)

Comparing the genetic composition of wild animals between geographic regions with distinct environments is common in evolutionary studies. However, genetic composition can also change through time in response to environmental changes but studies examining this are carried out less often. In this study, we characterise striped dolphin genetic composition in the Mediterranean Sea across both geography and time. We provide genotype data for 15 microsatellite loci and 919 bp of mtDNA control region, collected over 21 years across all main Mediterranean Sea basins. We investigated spatial genetic structure using both classical and Bayesian population structure methods, and compared it with temporal patterns of genetic change using time series statistics. We integrated the temporal datasets with known environmental pressures and data on social structure, to infer potential drivers of observed changes. Geographic analyses suggest weak differentiation for striped dolphin in the Mediterranean Sea, with evidence for a recent expansion. Temporal analyses show significant cyclical fluctuations in genetic composition over 21 years, which correspond well with recurrent morbillivirus epizootics. Similarly, social group composition shows changes in the relative number of juveniles and adults per group, and an overall increase in the number of adults per group relative to juveniles over the time period. We suggest that the observed changes in genetic and group composition could relate to specific dynamics of morbillivirus resistance. Overall, our study highlights the importance of tracking long term genetic variation, and the potential for this species as a model in studying genetic adaptation to environmental stress

The mesenchymal stem cell differentiation to osteoblasts is potentiate by the allosteric modulation of A2B adenosine receptors.

The A2B adenosine receptor (A2BAR) has been recently emerged as the major adenosine receptor involved in the mesenchymal stem cell differentiation to osteoblast and bone formation, highlighting this receptor as a new target in bone diseases. In the present study, we characterized a new 3-keto-indole-derivative (KI-7) as the first positive allosteric modulator (PAM) of the human A2B AR in mesenchymal stem cells (MSCs), and we investigated the potential activity of this compound as osteogenic agent. KI-7 was able to increase the effects of A2B AR of both endogenous and orthosteric agonists on the expression of osteogenic markers and on osteoblast mineralization. In the early phase of differentiation program, KI-7 significantly potentiated physiological and A2B agonist-mediated down-regulation of IL-6 release. Conversely, during the late stage of differentiation, when most of the cells have an osteoblast phenotype, KI-7 caused a sustained raise in IL-6 levels and an improvement in osteoblast viability. These data suggest that positive allosteric modulation of A2B AR not only favors MSC commitment to osteoblasts, but also ensures a greater survival of mature osteoblasts. Our study paves the way for a therapeutic use of selective positive allosteric modulators of A2B AR in the control of osteoblast differentiation, bone formation and fracture repair

Negative effects of a high tumour necrosis factor-α concentration on human gingival mesenchymal stem cell trophism: The use of natural compounds as modulatory agents

Background: Adult mesenchymal stem cells (MSCs) play a crucial role in the maintenance of tissue homeostasis and in regenerative processes. Among the different MSC types, the gingiva-derived mesenchymal stem cells (GMSCs) have arisen as a promising tool to promote the repair of damaged tissues secreting trophic mediators that affect different types of cells involved in regenerative processes. Tumour necrosis factor (TNF)-α is one of the key mediators of inflammation that could affect tissue regenerative processes and modify the MSC properties in in-vitro applications. To date, no data have been reported on the effects of TNF-α on GMSC trophic activities and how its modulation with anti-inflammatory agents from natural sources could modulate the GMSC properties. Methods: GMSCs were isolated and characterized from healthy subjects. The effects of TNF-α were evaluated on GMSCs and on the well-being of endothelial cells. The secretion of cytokines was measured and related to the modification of GMSC-endothelial cell communication using a conditioned-medium method. The ability to modify the inflammatory response was evaluated in the presence of Ribes nigrum bud extract (RBE). Results: TNF-α differently affected GMSC proliferation and the expression of inflammatory-related proteins (interleukin (IL)-6, IL-10, transforming growth factor (TGF)-β, and cyclooxygenase (COX)-2) dependent on its concentration. A high TNF-α concentration decreased the GMSC viability and impaired the positive cross-talk between GMSCs and endothelial cells, probably by enhancing the amount of pro-inflammatory cytokines in the GMSC secretome. RBE restored the beneficial effects of GMSCs on endothelial viability and motility under inflammatory conditions. Conclusions: A high TNF-α concentration decreased the well-being of GMSCs, modifying their trophic activities and decreasing endothelial cell healing. These data highlight the importance of controlling TNF-α concentrations to maintain the trophic activity of GMSCs. Furthermore, the use of natural anti-inflammatory agents restored the regenerative properties of GMSCs on endothelial cells, opening the way to the use and development of natural extracts in wound healing, periodontal regeneration, and tissue-engineering applications that use MSCs