Littérature scientifique sur le sujet « FM19G11 »

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. In ALS mice, neurodegeneration is associated with the proliferative restorative attempts of ependymal stem progenitor cells (epSPCs) that normally lie in a quiescent in the spinal cord. Thus, modulation of the proliferation of epSPCs may represent a potential strategy to counteract neurodegeneration. Recent studies demonstrated that FM19G11, a hypoxia-inducible factor modulator, induces epSPC self-renewal and proliferation. The aim of the study was to investigate whether FM19G11-loaded gold nanoparticles (NPs) can affect self-renewal and proliferation processes in epSPCs isolated from G93A-SOD1 mice at disease onset. We discovered elevated levels of SOX2, OCT4, AKT1, and AKT3, key genes associated with pluripotency, self-renewal, and proliferation, in G93A-SOD1 epSPCs at the transcriptional and protein levels after treatment with FM19G11-loaded NPs. We also observed an increase in the levels of the mitochondrial uncoupling protein (UCP) gene in treated cells. FM19G11-loaded NPs treatment also affected the expression of the cell cycle-related microRNA (miR)-19a, along with its target gene PTEN, in G93A-SOD1 epSPCs. Overall our findings establish the significant impact of FM19G11-loaded NPs on the cellular pathways involved in self-renewal and proliferation in G93A-SOD1 epSPCs, thus providing an impetus to the design of novel tailored approaches to delay ALS disease progression.

Barriergenesis is the process of maturation of the primary vascular network of the brain responsible for the establishment of the blood-brain barrier. It represents a combination of factors that, on the one hand, contribute to the process of migration and tubulogenesis of endothelial cells (angiogenesis), on the other hand, contribute to the formation of new connections between endothelial cells and other elements of the neurovascular unit. Astrocytes play a key role in barriergenesis, however, mechanisms of their action are still poorly examined. We have studied the effects of HIF-1 modulators acting on the cells of non-endothelial origin (neurons and astrocytes) on the development of the blood-brain barrier in vitro. Application of FM19G11 regulating expression of HIF-1 activity and GSI-1 suppressing gamma-secretase and/or proteasomal activity resulted in the elevated expression of thrombospondins and matrix metalloproteinases in the developing blood-brain barrier. However, it caused the opposite effect on VEGF expression thus promoting barrier maturation in vitro.

Previous studies have reported the up-regulation of the two-pore domain K+ channel K2P5.1 in the CD4+ T cells of patients with multiple sclerosis (MS) and rheumatoid arthritis (RA), as well as in a mouse model of inflammatory bowel disease (IBD). However, the mechanisms underlying this up-regulation remain unclear. Inflammation-associated hypoxia is involved in the pathogenesis of autoimmune diseases, such as IBD, MS, and RA, and T cells are exposed to a hypoxic environment during their recruitment from inflamed tissues to secondary lymphoid tissues. We herein investigated whether inflammation-associated hypoxia is attributable to the increased expression and activity of K2P5.1 in the splenic CD4+ T cells of chemically-induced IBD model mice. Significant increases in hypoxia-inducible factor (HIF)-1α transcripts and proteins were found in the splenic CD4+ T cells of the IBD model. In the activated splenic CD4+ T cells, hypoxia (1.5% O2) increased K2P5.1 expression and activity, whereas a treatment with the HIF inhibitor FM19G11 but not the selective HIF-2 inhibitor exerted the opposite effect. Hypoxia-exposed K2P5.1 up-regulation was also detected in stimulated thymocytes and the mouse T-cell line. The class III histone deacetylase sirtuin-1 (SIRT1) is a downstream molecule of HIF-1α signaling. We examined the effects of the SIRT1 inhibitor NCO-01 on K2P5.1 transcription in activated CD4+ T cells, and we found no significant effects on the K2P5.1 transcription. No acute compensatory responses of K2P3.1–K2P5.1 up-regulation were found in the CD4+ T cells of the IBD model and the hypoxia-exposed T cells. Collectively, these results suggest a mechanism for K2P5.1 up-regulation via HIF-1 in the CD4+ T cells of the IBD model.

La Sclerosi Laterale Amiotrofica (SLA) è una patologia neurodegenerativa progressiva in cui le disfunzioni della barriera emato-encefalica (BEE) contribuiscono alla patogenesi e limitano significativamente lo sviluppo di nuove terapie. Gli astrociti di pazienti affetti da forme familiari e sporadiche di SLA rilasciano fattori solubili implicati nel danno della BEE e nell’aumento della P-glicoproteina (P-gp), la principale pompa dell’efflusso di farmaci espressa dalle cellule endoteliali nel cervello, responsabile dei fenomeni di progressiva farmacoresistenza nella SLA. Recentemente è stato dimostrato che FM19G11, un nuovo composto chimico, limita l’attivazione astrocitaria in un modello di lesione del midollo spinale, riducendo la risposta cellulare allo stress. Lo scopo di questa tesi era esaminare gli effetti del trattamento con FM19G11 sugli astrociti reattivi di pazienti SLA a livello della BEE. Abbiamo quindi realizzato un modello in vitro di BEE derivato da cellule staminali umane pluripotenti indotte (hiPSC) e composto da cellule endoteliali controllo (EC) in co-coltura con astrociti ottenuti da iPSC di un soggetto controllo, un paziente SLA familiare con mutazione SOD1-A4V e un soggetto con SLA sporadica. Gli astrociti venivano trattati con FM19G11 per 48h. Parallelamente, le cellule endoteliali erano coltivate su una membrana porosa all’interno di inserti affinchè formassero il monostrato della barriera. Dopo 48h il terreno di coltura degli astrociti veniva sostituito con nuovo terreno e gli inserti con le cellule endoteliali (rappresentanti il “lato del circolo ematico” della BEE) venivano trasferite al di sopra delle colture di astrociti ( il “lato encefalico” della BBB). Le co-colture, che garantivano la contiguità tra i surnatanti evitando il contatto diretto tra le diverse popolazioni cellulari, venivano protratte per 5 giorni. La formazione e l’integrità del monostrato di cellule endoteliali veniva valutato tramite: immunocitochimica per Zonula Occludens-1 (ZO-1), una delle principali proteine costituenti le giunzioni serrate presenti tra le cellule endoteliali nel cervello; misurazione della resistenza elettrica trans-endoteliale (TEER); analisi della permeabilità di fluoresceina sodica (NaF), una molecola solubile in acqua che attraversa la barriera solo tramite diffusione paracellulare, indicatore quindi dell’integrità delle giunzioni tra le cellule endoteliali. Al fine di valutare la presenza del fenomeni di farmacoresistenza, abbiamo misurato l’attività di trasporto di P-gp tramite l’efflusso di Rodamina (Rh123), un substrato specifico di P-gp. Inoltre, l’espressione di P-gp da parte delle cellule endoteliali è stata analizzata tramite immunocitochimica e immunofissazione. Per valutare le vie molecolari sottese alla modulazione delle cellule endoteliali abbiamo calcolato il rapporto tra espressione nucleare e citoplasmatica del fattore NF-kB tramite analisi di immunocitochimica. Inoltre, abbiamo analizzato, tramite specifiche metodiche fluorometriche, l’effetto di FM19G11 sui possibili mediatori dell’attivazione di NF-kB rilasciati dagli astrociti reattivi da pazienti SLA, le specie reattive dell’ossigeno (ROS) e il glutammato. Rispetto alle co-colture composte da astrociti non trattati, le cellule endoteliali coltivate con gli astrociti SOD1-A4V a sporadici trattati con FM19G11 hanno mostrato: ì) una maggiore resistenza, congrua con una minore permeabilità, del monostrato; ii) una ridotta espressione e funzione di efflusso di P-gp; iii) una ridotta attivazione di NF-kB, in linea con la minore produzione di ROS e glutammato da parte degli astrociti SOD1-A4V e sporadici trattati con FM19G11. FM19G11 mantiene l’integrità della BEE e riduce la sovra espressione di P-gp ,diminuendo la reattività astrocitaria, pertanto potrebbe essere considerato per futuri approcci terapeutici combinatoriali per la terapia della SLA.
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease in which blood brain barrier (BBB) dysfunction contributes to the pathogenesis and significantly limits the development of successful therapies. Astrocytes from familial and sporadic ALS patients release soluble factors implicated in BBB injury and upregulation of P-glycoprotein (P-gp), the main multidrug efflux transporter in brain endothelial cells, responsible for progressive pharmacoresistance in ALS. Recently, FM19G11, a new chemical compound, was shown to counteract astrogliosis in a spinal cord injury model, decreasing cell stress response in the microenvironment. Specific objectives of this thesis were: i) to evaluate the potential effect of FM19G11 treatment on astrocyte-driven BBB dysruption and pharmacoresistance; ii) to identify ALS astrocyte molecular features targeted by FM19G11 compound. To this purpose, we set up a human induced pluripotent stem cells (hiPSCs)-derived BBB in vitro system composed of control iPS-endothelial cells (ECs), co-cultured with hiPSCs-derived astrocytes from control, familial SOD1-A4V and Sporadic ALS patients. Control and ALS-astrocytes were conditioned with FM19G11 500nM for 48h. In parallel, ECs were seeded on transwell porous membrane inserts, allowing to form the endothelial monolayer. After 48h, astrocyte culture medium was replaced with fresh medium, and ECs-inserts (representing the “blood side” of the BBB) were placed atop of the pre-treated astrocyte layer (representing the “brain side”). Co-cultures in the transwell culture system, which guaranteed supernatant interaction, avoiding cell contact, were maintained for 5 days. Formation and integrity of human iPSC endothelial monolayer were examined by: immunostaining for Zonula Occludens-1 (ZO-1), a major protein constituent of brain endothelial tight junctions (TJs); trans-endothelial electric resistance (TEER) assessment; sodium fluorescein (NaF) permeability assay, a water-soluble molecule that cross the BBB only through paracellular diffusion, thus being an indicator of endothelial cell-to-cell cohesion. In order to investigate pharmacoresistance occurrence, we determined P-gp transport activity by calculating Rhodamine 123 (Rh123), a P-gp specific substrate, efflux ratio. P-gP expression was assessed on endothelial monolayers by immunocytochemistry and western-blot analysis. To examine the molecular signaling underlying endothelial cells modulation, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) nuclear to cytoplasmic ratio was investigated by immunocytochemical expression analysis. Further, we examined FM19G11 effect on potential NF-kB upstream mediators released by reactive ALS astrocytes. Hence, astrocyte-derived reactive oxygen species (ROS) and glutamate levels in astrocyte conditioned medium were also measured by highly sensitive fluorometric approaches. Compared to co-cultures composed by untreated ALS-astrocytes, endothelial monolayers co-cultured with FM19G11-conditioned SOD1-A4V and sporadic astrocytes showed: i) an improvement in endothelial monolayer resistance, compatible with a decreased passive permeability across the BBB in vitro model; ii) a consistently lower P-gp expression and efflux activity; ììì) a reduced NF-kB activation, in line with the detected decreased ROS and glutamate production by FM19G11-treated SOD1-A4V and sporadic astrocytes. FM19G11 preserves BBB integrity, and restrains P-gP overexpression, by reducing cell stress factors released from familial SOD1-A4V and sporadic ALS astrocyte in a human-derived in vitro cell model. The outcome of our investigation suggests that FM19G11 is able to limit BBB dysfunction and could impact brain accessibility of therapeutics thus, it might be considered for future combinatorial therapeutic strategies for ALS.