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

α-Synuclein Heterocomplexes with β-Amyloid Are Increased in Red Blood Cells of Parkinson's Disease Patients and Correlate with Disease Severity

Neurodegenerative disorders (NDs) are characterized by abnormal accumulation/misfolding of specific proteins, primarily α-synuclein (α-syn), β-amyloid1-42(Aβ1-42) and tau, in both brain and peripheral tissues. In addition to oligomers, the role of the interactions of α-syn with Aβ or tau has gradually emerged. Nevertheless, despite intensive research, NDs have no accepted peripheral markers for biochemical diagnosis. In this respect, Red Blood Cells (RBCs) are emerging as a valid peripheral model for the study of aging-related pathologies. Herein, a small cohort (N= 28) of patients affected by Parkinson's disease (PD) and age-matched controls were enrolled to detect the content of α-syn (total and oligomeric), Aβ1-42and tau (total and phosphorylated) in RBCs. Moreover, the presence of α-syn association with tau and Aβ1-42was explored by co-immunoprecipitation/western blotting in the same cells, and quantitatively confirmed by immunoenzymatic assays. For the first time, PD patients were demonstrated to exhibit α-syn heterocomplexes with Aβ1-42and tau in peripheral tissues; interestingly, α-syn-Aβ1-42concentrations were increased in PD subjects with respect to healthy controls (HC), and directly correlated with disease severity and motor deficits. Moreover, total-α-syn levels were decreased in PD subjects and inversely related to their motor deficits. Finally, an increase of oligomeric-α-syn and phosphorylated-tau was observed in RBCs of the enrolled patients. The combination of three parameters (total-α-syn, phosphorylated-tau and α-syn-Aβ1-42concentrations) provided the best fitting predictive index for discriminating PD patients from controls. Nevertheless further investigations should be required, overall, these data suggest α-syn hetero-aggregates in RBCs as a putative tool for the diagnosis of PD

α-Synuclein Aggregates with β-Amyloid or Tau in Human Red Blood Cells: Correlation with Antioxidant Capability and Physical Exercise in Human Healthy Subjects

Neurodegenerative disorders (NDs) are characterized by abnormal accumulation/misfolding of specific proteins, primarily α-synuclein (α-syn), β-amyloid1–42 (Aβ), and tau, in both brain and peripheral tissue. In addition to homo-oligomers, the role of α-syn interactions with Aβ or tau has gradually emerged. The altered protein accumulation has been related to both oxidative stress and physical activity; nevertheless, no correlation among the presence of peripheral α-syn hetero-aggregates, antioxidant capacity, and physical exercise has been discovered as of yet. Herein, the content of α-syn, Aβ, tau, and of their heterocomplexes was determined in red blood cells (RBCs) of healthy subjects (sedentary and athletes). Such parameters were related to the extent of the antioxidant capability (AOC), a key marker of oxidative stress in aging-related pathologies, and to physical exercise, which is known to play an important preventive role in NDs and to modulate oxidative stress. Tau content and plasma AOC toward hydroxyl radicals were both reduced in older or sedentary subjects; in contrast, α-syn and Aβ accumulated in elderly subjects and showed an inverse correlation with both hydroxyl AOC and the level of physical activity. For the first time, α-syn heterocomplexes with Aβ or tau were quantified and demonstrated to be inversely related to hydroxyl AOC. Furthermore, α-syn/Aβ aggregates were significantly reduced in athletes and inversely correlated with physical activity level, independent of age. The positive correlation between antioxidant capability/physical activity and reduced protein accumulation was confirmed by these data and suggested that peripheral α-syn heterocomplexes may represent new indicators of ND-related protein misfolding

Modulatori "long-lasting" di MDM2: una strategia innovativa per indurre apoptosi irreversibile di cellule di glioblastoma umano.

Il glioblastoma multiforme (GBM) è il tumore cerebrale più aggressivo ed invasivo e con un elevatissimo tasso di mortalità, per cui rimane ad oggi una delle più importanti sfide in ambito oncologico. I pazienti affetti da glioblastoma hanno in media una sopravvivenza di 1-2 anni dalla diagnosi e comunemente, anche se trattati chirurgicamente e farmacologicamente, muoiono per l’insorgenza di recidive. La comparsa di recidive frequenti è correlata alla resistenza di tale tumore alla radioterapia e ai classici farmaci chemioterapici utilizzati, tra cui principalmente l’agente alchilante, Temozolomide. Per tali motivi, la ricerca si sta indirizzando fortemente negli ultimi anni verso lo studio dei meccanismi molecolari alla base della farmaco-resistenza di questo tumore al fine di ottimizzare lo sviluppo di innovative strategie terapeutiche. Un ruolo fondamentale nei meccanismi di apoptosi cellulare è svolto dalla proteina p53; oltre a promuovere l’espressione di specifici geni chiave nel controllo della proliferazione cellulare, la proteina p53 interviene nel ciclo cellulare e nella riparazione di danno al DNA ed è capace di innescare il processo apoptotico in seguito a danno genotossico. Nel GBM sono stati osservati sia una mutazione del gene codificante per p53, con conseguente perdita funzionale, sia una sovra-espressione della proteina Murine Double Minute-2 (MDM2), il principale regolatore negativo della stessa proteina p53. Molecole inibitrici dell’interazione p53/MDM2 rappresentano al momento un approccio farmacologico per il trattamento di questo tumore cerebrale. Inoltre, recenti scoperte nel campo della biologia molecolare hanno dimostrato che l'aberrazione di diverse vie di segnale è coinvolta nella patogenesi dei gliomi maligni, e hanno permesso di identificare nuovi bersagli per nuovi approcci terapeutici. Poiché queste vie di segnale intracellulare non funzionanti sono il punto di convergenza per diversi stimoli, la terapia multi-target sta diventando il modo preferito per lo sviluppo di terapie innovative e più efficienti. Oltre alla terapia multi-target, un approccio farmacologico efficace per il trattamento del glioblastoma è l’utilizzo di molecole capaci di legare proteine target in maniera irreversibile, tra cui ad esempio gli inibitori dei recettori tirosin chinasici di ultima generazione. È stato ipotizzato infatti che una molecola reversibile potrebbe non essere sufficiente per sostenere un effetto terapeutico nel tempo, favorendo l'attivazione di vie di segnale alternative che sfuggono l’azione dei farmaci e provocano fenomeni di farmaco-resistenza. Recentemente è stato suggerito, infatti, che gli effetti benefici di farmaci indirizzati verso altri target biologici e comunemente in uso per la cura di determinati stati patologici, possono essere attribuiti, più che all’alta affinità di legame del farmaco, alla lunga durata del complesso farmaco-bersaglio, parametro noto come “Residence Time”. Lo scopo della presente tesi è stato quello di valutare la potenziale attività anti-tumorale e caratterizzare il meccanismo d’azione, molecolare e cellulare, di una nuova molecola sintetica, EB148. Tale composto è stato sviluppato partendo dal derivato a struttura 2-fenilindol-3-ylglyoxyl-dipeptidica, EB54, che era già stato dimostrato essere in grado di riattivare la funzionalità di p53, tramite la dissociazione dal suo inibitore endogeno MDM2 (Murine Double Minute-2). Nelle cellule di glioblastoma umano (U87MG), tale composto è in grado di indurre blocco del ciclo cellulare ed apoptosi, portando quindi ad un blocco della vitalità/proliferazione cellulare. Partendo da EB54, il ligando irreversibile EB148 è stato sviluppato introducendo un gruppo elettrofilo in posizione 5 dell’anello indolico. Lo scopo dell’introduzione del sostituente isotiocianato è stato infatti quello di ottenere un ligando egualmente affine a MDM2, ma con un aumentato “residence time” al target biologico, in modo da prolungare e migliorare l’attività anti-proliferativa. Come primo step, è stata valutata la capacità della nuova molecola di dissociare MDM2 da p53 e il tempo di legame dei due ligandi al target MDM2. Il composto EB148 è risultato capace di dissociare efficacemente il complesso p53/MDM2, legandosi a MDM2 con un’affinità pari a 6.81 ± 0.79 nM, valore comparabile con quello dell’analogo reversibile (IC50=11.65 ± 0.49 nM). Per verificare il meccanismo d’azione covalente del composto, inoltre, sono stati svolti studi in cinetica di dissociazione da MDM2. I risultati ottenuti hanno dimostrato che, in presenza di EB148, l’inibizione del complesso p53/MDM2 viene mantenuta costante anche dopo diluizione del campione. Al contrario, la diluizione dei campioni ha invece diminuito progressivamente il legame a MDM2 di EB54, confermando la natura reversibile dell’interazione con MDM2. La riattivazione della funzionalità di p53 da parte di EB148 è stata verificata valutando sia i livelli trascrizionali dei geni target di p53, sia i livelli di espressione della proteina p53 stessa. I risultati hanno confermato che EB54 induce un aumento significativo di mRNA dei geni target di p53 e un significativo accumulo di tale proteina, ma con effetti che non persistono dopo rimozione del ligando. Al contrario, EB148 è in grado di attivare p53 in modo sostenuto nel tempo, mantenendo livelli di proteina significativamente più alti rispetto al controllo anche dopo rimozione del ligando. Successivamente, è stato valutato il coinvolgimento delle MAPK (Mitogen-activated protein kinase) ERK1/2 negli effetti mediati dai composti. Il modello cinetico di attivazione delle ERK è infatti il pre-requisito che regola i risultati biologici finali di queste proteine segnale. Mentre un’attivazione temporanea di queste chinasi è comunemente associata con un effetto proliferativo, la loro fosforilazione sostenuta nel tempo innesca le vie di segnale di morte cellulare. Mentre il composto reversibile non induce nessuna significativa stimolazione delle ERK1/2, EB148 induce una significativa fosforilazione delle ERK e il segnale rimane elevato fino a 8 ore di trattamento. Inoltre, anche dopo un wash-out, il composto irreversibile continua ad attivare queste chinasi. Tale segnale è risultato essere mediato da p53. Infine, i composti sono stati valutati per i loro effetti sulla proliferazione/vitalità cellulare. Entrambi i ligandi inibiscono la vitalità cellulare delle cellule U87MG con valori di IC50 paragonabili. A differenza di quanto visto con EB54, la rimozione di EB148 non ha comportato un recupero della normale crescita delle cellule rimaste, suggerendo la compromissione dei meccanismi che regolano la progressione cellulare. I risultati, quindi, hanno dimostrato che la molecola risulta essere attiva a livello del complesso p53/MDM2 e le proprietà irreversibili di associazione possono rappresentare una strategia utile per guidare le cellule di GBM verso un’apoptosi irreversibile, superando gli effetti di durata limitata tipici dei chemioterapici classici

High Adenosine Extracellular Levels Induce Glioblastoma Aggressive Traits Modulating the Mesenchymal Stromal Cell Secretome

Glioblastoma is an aggressive, fast-growing brain tumor influenced by the composition of the tumor microenvironment (TME) in which mesenchymal stromal cell (MSCs) play a pivotal role. Adenosine (ADO), a purinergic signal molecule, can reach up to high micromolar concentrations in TME. The activity of specific adenosine receptor subtypes on glioma cells has been widely explored, as have the effects of MSCs on tumor progression. However, the effects of high levels of ADO on glioma aggressive traits are still unclear as is its role in cancer cells-MSC cross-talk. Herein, we first studied the role of extracellular Adenosine (ADO) on isolated human U343MG cells as a glioblastoma cellular model, finding that at high concentrations it was able to prompt the gene expression of Snail and ZEB1, which regulate the epithelial–mesenchymal transition (EMT) process, even if a complete transition was not reached. These effects were mediated by the induction of ERK1/2 phosphorylation. Additionally, ADO affected isolated bone marrow derived MSCs (BM-MSCs) by modifying the pattern of secreted inflammatory cytokines. Then, the conditioned medium (CM) of BM-MSCs stimulated with ADO and a co-culture system were used to investigate the role of extracellular ADO in GBM–MSC cross-talk. The CM promoted the increase of glioma motility and induced a partial phenotypic change of glioblastoma cells. These effects were maintained when U343MG cells and BM-MSCs were co-cultured. In conclusion, ADO may affect glioma biology directly and through the modulation of the paracrine factors released by MSCs overall promoting a more aggressive phenotype. These results point out the importance to deeply investigate the role of extracellular soluble factors in the glioma cross-talk with other cell types of the TME to better understand its pathological mechanisms

Signalling pathways in Glioblastoma as target for innovative therapy

Glioblastoma (GBM) is an aggressive and invasive brain tumor. The first line treatment usually is the surgical resection, followed by radiotherapy and the administration of the alkylating agent Temozolomide. Significant intra- and inter-tumor heterogeneity have been associated with aberrations in different intracellular pathways and a peculiar tumor microenvironment (TME) that contributes to GBM aggressiveness. In GBM, tumor cells can escape from apoptosis because there is an overexpression of anti-apoptotic proteins and an inhibition of pro-apoptotic ones, like Bax. The tumor progression could be due also to the deregulation of the oncosuppressor protein p53. The overexpression of p53 inhibitor, MDM2, often occurs in GBM promoting its proliferation. The reactivation of p53 endogenous function can represent an important target in the development of effective GBM treatment. The failure of GBM treatment is also due to the presence of a peculiar TME. Tumor cells regulate the function of cellular and non-cellular components through complex signalling pathways and could use the non-malignant cells to work for their benefit. Adenosine is one of the main immunomodulatory mediators involved in tumor development. Herein, we deeply investigated the aberrant intracellular signalling pathways involved in GBM pathogenesis and how the modulation of these pathways could prevent GBM aggressiveness

Human gingival mesenchymal stem cell trophism is modulated by inflammatory microenvironment: effects of ribes nigrum bud extract

Adult mesenchymal stem cells (MSCs) play a crucial role in the maintenance of tissue homeostasis and in promoting regenerative processes. Among the different MSC types, the gingival mesenchymal stem cells (GMSCs) have arisen as a promising tool to promote the repair of damaged tissues secreting trophic, regeneration-promoting mediators. TNF-α is one of the key mediators of inflammation that could affect tissue regenerative processes and modify the MSC properties in in vitro application. Herein, we investigated 1) the effects of TNF-alpha on GMSC trophism and 2) the ability of Ribes Nigrum bud extract (RBE) to modulate the effect of this cytokine on GMSC properties. GMSC were isolated and characterized from health subjects. TNF-α affected GMSC proliferation and the expression of inflammatory-related protein (IL-6, IL-10, TGF-β, and COX-2) in dependence on its concentration. A high TNF-α concentration decreased the GMSC viability and impaired the trophic effect of GMSCs on endothelial cells, likely by enhancing the amount of pro-inflammatory mediators in GMSC secretome. GMSC incubation with RBE changed secretoma cell composition so restoring the GMSC beneficial effects on endothelial viability and motility. These results demonstrated that a high TNF-α concentration, as occurred under chronic inflammatory conditions, decreased the GMSC well-being and alter their trophic activity impairing GMSC-endothelial cell communication. These data highlight that the control of inflammatory microenvironment is crucial to guarantee MSC-driven reparative processes. Furthermore, the use of natural anti-inflammatory agents restored the GMSC regenerative properties on endothelial cells opening the way to the use and the development of natural extracts in wound healing, periodontal regeneration and tissue engineering application that use MSC

Influence of physical exercise on β-amyloid, α-synuclein and tau accumulation: an in vitro model of oxidative stress in human red blood cells.

A common pathological feature of neurodegenerative disorders (NDs), such as Alzheimer's (AD) and Parkinson's (PD) diseases, is the abnormal accumulation and misfolding of specific proteins, primarily α-synuclein (α-syn), β-amyloid1-42 (Aβ) and tau, in brain and in peripheral tissues too. Oxidative stress has been proved to be involved in NDs at various levels and, in particular, in such protein alterations, on the contrary physical activity is emerging as a counteracting factor in NDs. In the present work, the content of Aβ, α-syn and tau in red blood cells (RBCs) derived from ten endurance athletes (ATHL) and ten sedentary volunteers (SED) were compared before and after in vitrooxidative stress treatment. Total Aβ, α-syn and tau were quantified in RBCs (isolated from the subjects) by immunoenzymatic assays. Oxidative stress was induced by in vitro H₂O₂ administration to RBCs. H₂O₂ treatment was confirmed to significantly enhance ROS accumulation in RBCs. Total Aβ content in RBCs was lower in the ATHL subgroup with respect to the SED one. In the SED subgroup, but not in the ATHL one, total Aβ levels were increased by oxidative stress. Total α-syn content was lower in the ATHL subgroup with respect to the SED one and α-syn levels were increased by oxidative stress in both subgroups, with the percentage of increase higher in SED. Total tau content was comparable in both ATHL and SED and it was not affected by oxidative stress. Our data confirm previous findings evidencing that both oxidative stress and sedentary style contribute to aberrant folding and accumulation of NDs-related proteins, pointing to the importance of both anti-oxidant therapies and exercising in the prevention and treating of such diseases

New insights on the human bone-marrow and gingival mesenchymal stem cell responses to senescence induction

Bone-marrow mesenchymal stem cells (BMSCs) has been widely used in regenerative medicine, even if they rapidly undergo to senescence phenomena limiting their use in vitro. A promising alternative to BMSCs are the gingival MSCs (GMSCs) for which the susceptibility to senescence induction is still unclear. Herein, we investigated the functional responses of BMSCs and GMCs to two different senescence cellular models that were set up utilizing sub-lethal concentrations of hydroxyurea (HU) and hydrogen peroxide (H2O2). Despite these two models effectively induced BMSC and GMSC cells to undergo cellular senescence, the age-related phenotypic changes (SA-β-gal staining) were significantly lower in GMSCs. By evaluating the expression of different senescence-related genes, including p53, p21 and p16INK4α, we demonstrated GMSCs maintain a higher proliferation rate and result more resistant to HU and H2O2 treatment. BMSC were more prone to apoptotic phenomena and showed higher levels of intracellular reactive oxygen species. In conclusion, GMSCs are more prone to contrast the senescence induction confirming these staminal cell populations as an attractive tool in regenerative medicin

Dual inhibition of PDK1 and Aurora Kinase A: an effective strategy to induce differentiation and apoptosis of human glioblastoma multiforme stem cells

The poor prognosis of Glioblastoma Multiforme (GBM) is mainly attributed to drug resistance mechanisms and to the existence of a subpopulation of glioma stem cells (GSCs). Multi-target compounds able to both affect different deregulated pathways and the GSC subpopulation could escape tumour resistance, and most importantly, eradicate the stem cell reservoir. In this respect, the simultaneous inhibition of Phosphoinositide-dependent kinase-1 (PDK1) and Aurora Kinase A (AurA), each one playing a pivotal role in cellular survival/migration/differentiation, could represent an innovative strategy to overcome GBM resistance and recurrence. Herein, the cross-talk between these pathways was investigated, using the single-target reference compounds MP7 (PDK1 inhibitor) and Alisertib (AurA inhibitor). Furthermore, a new ligand, SA16, was identified for its ability to inhibit the PDK1 and the AurA pathways at once, thus proving to be a useful tool for the simultaneous inhibition of the two kinases. SA16 blocked GBM cell proliferation, reduced tumour invasiveness, and triggered cellular apoptosis. Most importantly, the AurA/PDK1 blocker showed an increased efficacy against GSCs, inducing their differentiation and apoptosis. To the best of our knowledge, this is the first report on combined targeting of PDK1 and AurA. This drug represents an attractive multi-target lead scaffold for the development of new potential treatments for GBM and GSCs