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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 O, 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 molecu

Aberrant hexanucleotide repeat expansions in C9orf72 are the most common genetic change underlying amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). RNA transcripts containing these expansions undergo repeat-associated non-ATG translation (RAN-T) to form five dipeptide repeat proteins (DPRs). DPRs are found as aggregates throughout the CNS of C9orf72-ALS/FTD patients, and some cause degeneration when expressed in vitro in neuronal cultures and in vivo in animal models. The spread of characteristic disease-related proteins drives the progression of pathology in many neurodegenerative diseases. While DPR toxic mechanisms continue to be investigated, the potential for DPRs to spread has yet to be determined. Using different experimental cell culture platforms, including spinal motor neurons derived from induced pluripotent stem cells from C9orf72-ALS patients, we found evidence for cell-to-cell spreading of DPRs via exosome-dependent and exosome-independent pathways, which may be relevant to disease.