Ruolo della chinasi GRK2 nella desensitizzazione del recettore GPR17

La scopo di questa tesi è stato quello di studiare il ruolo della GRK, in particolare della GRK2, nei processi di desensitizzazione del GPR17. Questo GPCR è un recettore dualistico, che risponde a due classi di molecole: ai ligandi uridinici (quali UDP, UDP-glucosio) e ai cisteinil leucotrieni (LTC4, LTD4, LTE4), con una affinità nell’ordine del micromolare e nanomolare, rispettivamente. In condizioni patologiche, quali ischemia cerebrale e lesioni al midollo spinale, sono stati osservati profondi cambiamenti nel profilo di espressione spazio-temporale di questo recettore a partire dal sito della lesione: il GPR17 funziona infatti come “sensore” attivato dal danno, inducendo, nelle prime fasi, la morte cellulare nel core della lesione, e promuovendo, in tempi successivi, processi riparativi e di ripresa della funzione tissutale. Studi recenti dimostrano che l'espressione del GPR17 aumenta durante il differenziamento degli OPC a pre-oligodendrociti, e si riduce successivamente durante la transizione a cellule mature, producenti mielina; a stadi avanzati di differenziamento sembra essere necessaria una drastica diminuzione dell’espressione del recettore GPR17 affinchè le cellule possano completare la maturazione. Sulla base di questi dati è stato presupposto che, affinchè si completi la maturazione dei pre-oligodendrociti, questo GPCR debba andare incontro a fenomeni di desensitizzazione (ovvero perdita della funzionalità in seguito a stimolazione prolungata del recettore), internalizzazione e down-regulation. La desensitizzazione di un GPCR implica, in genere, la fosforilazione del recettore su residui amminoacidici conservati, come serina e treonina, ad opera di chinasi specifiche, (dette GRK) e la conseguente internalizzazione del complesso recettore-ligando. Questo processo va ad aumentare l’affinità del recettore per una proteina citoplasmatica, denominata ß-arrestina, che, una volta legatasi al recettore fosforilato, ne permette l’internalizzazione. Tra le diverse GRK, il ruolo centrale della GRK2 in molti pathway cellulari, suggerisce che l’alterata espressione di questa proteina e/o la sua attività possano avere importanti effetti sulle vie di segnale intracellulari

Trazodone treatment protects neuronal-like cells from inflammatory insult by inhibiting NF-κB, p38 and JNK

Growing evidence suggests that alterations of the inflammatory/immune system contribute to the pathogenesis of major depression and that inflammatory processes may influence the antidepressant treatment response. Depressed patients exhibit increased levels of inflammatory markers in both the periphery and brain, and high co-morbidity exists between depression and diseases associated with inflammatory alterations. Trazodone (TDZ) is a triazolopyridine derivative that belongs to the class of serotonin receptor antagonists and reuptake inhibitors. Although the trophic and protective properties of classic antidepressants have extensively been exploited, the effects of TDZ remain to be fully elucidated. In this study, the pharmacological activities of TDZ on human neuronal-like cells were investigated under both physiological and inflammatory conditions. An in vitro inflammatory model was established using lipopolysaccharide (LPS) and tumour necrosis factor-α (TNF-α), which efficiently mimic the stress-related changes in neurotrophic and pro-inflammatory genes.Our results showed that TDZ significantly increased the mRNA expression of both brain-derived nerve factor (BDNF) and cAMP response element-binding protein (CREB) and decreased the cellular release of the pro-inflammatory cytokine interferon gamma (IFN-γ) in neuronal-like cells.In contrast, neuronal cell treatment with LPS and TNF-α decreased the expression of CREB and BDNF and increased the expression of nuclear factor kappa B (NF-κB), a primary transcription factor that functions in inflammatory response initiation. Moreover, the two agents induced the release of pro-inflammatory cytokines (. i.e., interleukin-6 and IFN-γ) and decreased the production of the anti-inflammatory cytokine interleukin-10. TDZ pre-treatment completely reversed the decrease in cell viability and counteracted the decrease in BDNF and CREB expression mediated by LPS-TNF-α. In addition, the production of inflammatory mediators was inhibited, and the release of interleukin-10 was restored to control levels.Furthermore, the intracellular signalling mechanism regulating TDZ-elicited effects was specifically investigated. TDZ induced extracellular signal-regulated kinase (ERK) phosphorylation and inhibited constitutive p38 activation. Moreover, TDZ counteracted the activation of p38 and c-Jun NH2-terminal kinase (JNK) elicited by LPS-TNF-α, suggesting that the neuro-protective role of TDZ could be mediated by p38 and JNK.Overall, our results demonstrated that the protective effects of TDZ under inflammation in neuronal-like cells function by decreasing pro-inflammatory signalling and by enhancing anti-inflammatory signalling

Lactate dehydrogenase-A inhibition induces human glioblastoma multiforme stem cell differentiation and death

Therapies that target the signal transduction and metabolic pathways of cancer stem cells (CSCs) are innovative strategies to effectively reduce the recurrence and significantly improve the outcome of glioblastoma multiforme (GBM). CSCs exhibit an increased rate of glycolysis, thus rendering them intrinsically more sensitive to prospective therapeutic strategies based on the inhibition of the glycolytic pathway. The enzyme lactate dehydrogenase-A (LDH-A), which catalyses the interconversion of pyruvate and lactate, is up-regulated in human cancers, including GBM. Although several papers have explored the benefits of targeting cancer metabolism in GBM, the effects of direct LDH-A inhibition in glial tumours have not yet been investigated, particularly in the stem cell subpopulation. Here, two representative LDH-A inhibitors (NHI-1 and NHI-2) were studied in GBM-derived CSCs and compared to differentiated tumour cells. LDH-A inhibition was particularly effective in CSCs isolated from different GBM cell lines, where the two compounds blocked CSC formation and elicited long-lasting effects by triggering both apoptosis and cellular differentiation. These data demonstrate that GBM, particularly the stem cell subpopulation, is sensitive to glycolytic inhibition and shed light on the therapeutic potential of LDH-A inhibitors in this tumour type

The ubiquitin ligase Mdm2 controls oligodendrocyte maturation by intertwining mTOR with G protein-coupled receptor kinase 2 in the regulation of GPR17 receptor desensitization

During oligodendrocyte precursor cell (OPC) differentiation, defective control of the membrane receptor GPR17 has been suggested to block cell maturation and impair remyelination under demyelinating conditions. After the immature oligodendrocyte stage, to enable cells to complete maturation, GPR17 is physiologically down-regulated via phosphorylation/desensitization by G protein-coupled receptor kinases (GRKs); conversely, GRKs are regulated by the "mammalian target of rapamycin" mTOR. However, how GRKs and mTOR are connected to each other in modulating GPR17 function and oligodendrogenesis has remained elusive. Here we show, for the first time, a role for Murine double minute 2 (Mdm2), a ligase previously involved in ubiquitination/degradation of the onco-suppressor p53 protein. In maturing OPCs, both rapamycin and Nutlin-3, a small molecule inhibitor of Mdm2-p53 interactions, increased GRK2 sequestration by Mdm2, leading to impaired GPR17 down-regulation and OPC maturation block. Thus, Mdm2 intertwines mTOR with GRK2 in regulating GPR17 and oligodendrogenesis and represents a novel actor in myelination

Long lasting MDM2/Translocator protein modulator: a new strategy for irreversible apoptosis of human glioblastoma cells

The development of multi-target drugs and irreversible modulators of deregulated signalling proteins is the major challenge for improving glioblastoma multiforme (GBM) treatment. Reversible single-target drugs are not sufficient to sustain a therapeutic effect over time and may favour the activation of alternative signalling pathways and the onset of resistance phenomena. Thus, a multi-target compound that has a long-lasting mechanism of action might have a greater and longer life span of anti-proliferative activity. Recently, a dual-target indol-3ylglyoxyldipeptide derivative, designed to bind to the Translocator Protein (TSPO) and reactivate p53 function via dissociation from its physiological inhibitor, murine double minute 2 (MDM2), has been developed as a potent GBM pro-apoptotic agent. In this study, this derivative was chemically modified to irreversibly bind MDM2 and TSPO. The new compound elicited a TSPO-mediated mitochondrial membrane dissipation and restored p53 activity, triggering a long-lasting apoptosis of GBM cells. These effects were sustained over time, involved a stable activation of extracellular signal regulated kinases and were specifically observed in cancer cells, in which these protein kinases are deregulated. Dual-targeting and irreversible binding properties combined in the same molecule may represent a useful strategy to overcome the time-limited effects elicited by classical chemotherapies

Combined inhibition of AKT/mTOR and MDM2 enhances Glioblastoma Multiforme cell apoptosis and differentiation of cancer stem cells

The poor prognosis of Glioblastoma Multiforme (GBM) is due to a high resistance to conventional treatments and to the presence of a subpopulation of glioma stem cells (GSCs). Combination therapies targeting survival/self-renewal signals of GBM and GSCs are emerging as useful tools to improve GBM treatment. In this context, the hyperactivated AKT/mammalian target of the rapamycin (AKT/mTOR) and the inhibited wild-type p53 appear to be good candidates. Herein, the interaction between these pathways was investigated, using the novel AKT/mTOR inhibitor FC85 and ISA27, which re-activates p53 functionality by blocking its endogenous inhibitor murine double minute 2 homologue (MDM2). In GBM cells, FC85 efficiently inhibited AKT/mTOR signalling and reactivated p53 functionality, triggering cellular apoptosis. The combined therapy with ISA27 produced a synergic effect on the inhibition of cell viability and on the reactivation of p53 pathway. Most importantly, FC85 and ISA27 blocked proliferation and promoted the differentiation of GSCs. The simultaneous use of these compounds significantly enhanced GSC differentiation/apoptosis. These findings suggest that FC85 actively enhances the downstream p53 signalling and that a combination strategy aimed at inhibiting the AKT/mTOR pathway and re-activating p53 signalling is potentially effective in GBM and in GSCs

Discovery of MDM2-p53 and MDM4-p53 protein-protein interactions small molecule dual inhibitors

MDM2 and MDM4 are key negative regulators of p53, an important protein involved in several cell processes (e. g. cell cycle and apoptosis). Not surprisingly, the p53 tumor suppressor function is inactivated in tumors over -expressing these two proteins. Therefore, both MDM2 and MDM4 are considered important therapeutic targets for an effective reactivation of the p53 function. Herein, we present our studies on the development of spi-ropyrazoline oxindole small molecules able to inhibit MDM2/4-p53 protein-protein interactions (PPIs). Twenty-seven potential spiropyrazoline oxindole dual inhibitors were prepared based on in silico structural optimization studies of a hit compound with MDM2 and MDM4 proteins. The antiproliferative activity of the target com-pounds was evaluated in cancer cell lines harboring wild-type p53 and overexpressing MDM2 and/or MDM4. The most active compounds in SJSA-1 cells, 2q and 3b, induce cell death via apoptosis and control cell growth by targeting the G0/G1 cell cycle checkpoint in a concentration-dependent manner. The ability of the five most active spiropyrazoline oxindoles in dissociating p53 from MDM2 and MDM4 was analyzed by an immu-noenzymatic assay. Three compounds inhibited MDM2/4-p53 PPIs with IC50 values in the nM range, while one compound inhibited more selectively the MDM2-p53 PPI over the MDM4-p53 PPI. Collectively, these results show: i) 3b may serve as a valuable lead for obtaining selective MDM2-p53 PPI inhibitors and more efficient anti-osteosarcoma agents; ii) 2a, 2q and 3f may serve as valuable leads for obtaining dual MDM2/4 inhibitors and more effective p53 activators

Trazodone regulates neurotrophic/growth factors, mitogen-activated protein kinases and lactate release in human primary astrocytes

Background: In the central nervous system, glial cells provide metabolic and trophic support to neurons and respond to protracted stress and insults by up-regulating inflammatory processes. Reactive astrocytes and microglia are associated with the pathophysiology of neuronal injury, neurodegenerative diseases and major depression, in both animal models and human brains. Several studies have reported clear anti-inflammatory effects of anti-depressant treatment on astrocytes, especially in models of neurological disorders. Trazodone (TDZ) is a triazolopyridine derivative that is structurally unrelated to other major classes of antidepressants. Although the molecular mechanisms of TDZ in neurons have been investigated, it is unclear whether astrocytes are also a TDZ target. Methods: The effects of TDZ on human astrocytes were investigated in physiological conditions and following inflammatory insult with lipopolysaccharide (LPS) and tumour necrosis factor-aα (TNF-aα). Astrocytes were assessed for their responses to pro-inflammatory mediators and cytokines, and the receptors and signalling pathways involved in TDZ-mediated effects were evaluated. Results: TDZ had no effect on cell proliferation, but it decreased pro-inflammatory mediator release and modulated trophic and transcription factor mRNA expression. Following TDZ treatment, the AKT pathway was activated, whereas extracellular signal-regulated kinase and c-Jun NH2-terminal kinase were inhibited. Most importantly, a 72-h TDZ pre-treatment before inflammatory insult completely reversed the anti-proliferative effects induced by LPS-TNF-aα. The expression or the activity of inflammatory mediators, including interleukin-6, c-Jun NH2-terminal kinase and nuclear factor ΚB, were also reduced. Furthermore, TDZ affected astrocyte metabolic support to neurons by counteracting the inflammation-mediated lactate decrease. Finally, TDZ protected neuronal-like cells against neurotoxicity mediated by activated astrocytes. These effects mainly involved an activation of 5-HT1A and an antagonism at 5-HT2A/C serotonin receptors. Fluoxetine, used in parallel, showed similar final effects nevertheless it activates different receptors/intracellular pathways. Conclusions: Altogether, our results demonstrated that TDZ directly acts on astrocytes by regulating intracellular signalling pathways and increasing specific astrocyte-derived neurotrophic factor expression and lactate release. TDZ may contribute to neuronal support by normalizing trophic and metabolic support during neuroinflammation, which is associated with neurological diseases, including major depression