Investigazione del ruolo biologico di reelin, proteina extracellulare cerebrale, nelle cellule staminali neurali murine dell’adulto

Le cellule staminali neurali sono presenti, nel cervello dei mammiferi, in quelle aree dove la neurogenesi è mantenuta durante tutta la vita dell’organismo. Una caratteristica importante riguarda la loro abilità di proliferare e migrare nella materia cerebrale attratte dai siti di danno indotto, per esempio, da numerose malattie degenerative e dai tumori cerebrali. Il punto focale del mio dottorato è stato quello di studiare il ruolo di Reelin, proteina della matrice extracellulare fortemente implicata nello sviluppo cerebrale, nelle cellule staminali neurali murine. Mettendo a confronto le cellule “selvatiche” con quelle estratte da topi reeler, caratterizzate dalla mutazione negativa spontanea nel gene, ho osservato che l’assenza di Reelin rallenta la proliferazione delle cellule staminali neurali, così come la formazione delle neurosfere, aggregati sferici in sospensione tipici della crescita in vitro di queste cellule. Inoltre ho dimostrato un ruolo della proteina nel potenziale di differenziazione, favorendo la neuronogenesi in vitro senza modificarne la gliogenesi. Infine ho riscontrato l’incapacità delle cellule reeler di migrare nello stato aggregato chiamato “a catena”, la modalità di migrazione caratteristica delle cellule staminali neurali in vivo. Tutti questi effetti sono parzialmente recuperati dalla somministrazione della proteina esogena alle cellule reeler o con la diretta ingegnerizzazione per ripristinarne l’espressione endogena. Le conclusioni ricavate dal mio studio assegnano a Reelin un ruolo chiave nella biologia delle cellule staminali neurali adulte, intervenendo nella proliferazione, nel differenziamento e nella migrazione, le tre principali caratteristiche che guidano il processo di rigenerazione del tessuto danneggiato.In the adult mammalian brain, multipotential neural stem cells persist throughout life in those areas where neurogenesis is maintained. A distinctive trait of these cells is their ability to self-renew and to migrate through brain matter to sites of injury, such as those of occurrence of gliomas and neurodegenerative deseases. The aim of my doctorate study was the role of Reelin, an extracellular matrix protein deeply involved in brain development, in newborn mouse neural stem cells (NSCs). By comparing wild type and Reelin knock out reeler stem cells, I show that the absence of Reelin negatively affects proliferation and neurosphere-forming ability, as well as in vitro neuronal differentiation potential. Notably, reeler NSCs are not able to migrate in chains, a migration mode typical of neural precursors homing to injury sites in adult CNS. All these effects are partially rescued by ectopic Reelin supplementation. Overall, my results indicate that Reelin affects all three major features of post-natal NSCs, and that it is required for the proper homing of NSCs to injury sites in adult brain

miR-221 and miR-222 Expression Affects the Proliferation Potential of Human Prostate Carcinoma Cell Lines by Targeting p27Kip1

MicroRNAs are short regulatory RNAs that negatively modulate protein expression at a post-transcriptional level and are deeply involved in the pathogenesis of several types of cancers. Here we show that miR-221 and miR-222, encoded in tandem on chromosome X, are overexpressed in the PC3 cellular model of aggressive prostate carcinoma, as compared with LNCaP and 22Rv1 cell line models of slowly growing carcinomas. In all cell lines tested, we show an inverse relationship between the expression of miR-221 and miR-222 and the cell cycle inhibitor p27(Kip1). We recognize two target sites for the microRNAs in the 3' untranslated region of p27 mRNA, and we show that miR-221/222 ectopic overexpression directly results in p27 down-regulation in LNCaP cells. In those cells, we demonstrate that the ectopic overexpression of miR-221/222 strongly affects their growth potential by inducing a G(1) to S shift in the cell cycle and is sufficient to induce a powerful enhancement of their colony-forming potential in soft agar. Consistently, miR-221 and miR-222 knock-down through antisense LNA oligonucleotides increases p27(Kip1) in PC3 cells and strongly reduces their clonogenicity in vitro. Our results suggest that miR-221/222 can be regarded as a new family of oncogenes, directly targeting the tumor suppressor p27(Kip1), and that their overexpression might be one of the factors contributing to the oncogenesis and progression of prostate carcinoma through p27(Kip1) down-regulation

Retinal pigment epithelium degeneration caused by aggregation of PRPF31 and the role of HSP70 family of proteins

Background Mutations in pre-mRNA splicing factor PRPF31 can lead to retinitis pigmentosa (RP). Although the exact disease mechanism remains unknown, it has been hypothesized that haploinsufficiency might be involved in the pathophysiology of the disease. Methods In this study, we have analyzed a mouse model containing the p.A216P mutation in Prpf31 gene. Results We found that mutant Prpf31 protein produces cytoplasmic aggregates in the retinal pigment epithelium and decreasing the protein levels of this splicing factor in the nucleus. Additionally, normal protein was recruited in insoluble aggregates when the mutant protein was overexpressed in vitro. In response to protein aggregation, Hspa4l is overexpressed. This member of the HSP70 family of chaperones might contribute to the correct folding and solubilization of the mutant protein, allowing its translocation to the nucleus. Conclusions Our data suggests that a mechanism haploinsufficiency and dominant-negative is involved in retinal degeneration due to mutations in PRPF31. HSP70 over-expression might be a new therapeutic target for the treatment of retinal degeneration due to PRPF31 mutations.This project has been financed through a) The ISCIII (Miguel Servet-I, 2015), co-financed by the European Regional Development Fund (ERDF), No CP15/00071. b) The European Union’s Horizon 2020 research and innovation program, under grant agreement No 634479. c) Regional Ministry of Economy, Innovation and Science of the Junta de Andalucía, No P09-CTS-04967.info:eu-repo/semantics/publishedVersio

Organoid models of fibrolamellar carcinoma mutations reveal hepatocyte transdifferentiation through cooperative BAP1 and PRKAR2A loss

Fibrolamellar carcinoma (FLC) is a lethal primary liver cancer, affecting young patients in absence of chronic liver disease. Molecular understanding of FLC tumorigenesis is limited, partly due to the scarcity of experimental models. Here, we CRISPR-engineer human hepatocyte organoids to recreate different FLC backgrounds, including the predominant genetic alteration, the DNAJB1-PRKACA fusion, as well as a recently reported background of FLC-like tumors, encompassing inactivating mutations of BAP1 and PRKAR2A. Phenotypic characterizations and comparisons with primary FLC tumor samples revealed mutant organoid-tumor similarities. All FLC mutations caused hepatocyte dedifferentiation, yet only combined loss of BAP1 and PRKAR2A resulted in hepatocyte transdifferentiation into liver ductal/progenitor-like cells that could exclusively grow in a ductal cell environment. BAP1-mutant hepatocytes represent primed cells attempting to proliferate in this cAMP-stimulating environment, but require concomitant PRKAR2A loss to overcome cell cycle arrest. In all analyses, DNAJB1-PRKACAfus organoids presented with milder phenotypes, suggesting differences between FLC genetic backgrounds, or for example the need for additional mutations, interactions with niche cells, or a different cell-of-origin. These engineered human organoid models facilitate the study of FLC

Human fetal brain self-organizes into long-term expanding organoids

Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.</p

Engineered human hepatocyte organoids enable CRISPR-based target discovery and drug screening for steatosis

The lack of registered drugs for nonalcoholic fatty liver disease (NAFLD) is partly due to the paucity of human-relevant models for target discovery and compound screening. Here we use human fetal hepatocyte organoids to model the first stage of NAFLD, steatosis, representing three different triggers: free fatty acid loading, interindividual genetic variability (PNPLA3 I148M) and monogenic lipid disorders (APOB and MTTP mutations). Screening of drug candidates revealed compounds effective at resolving steatosis. Mechanistic evaluation of effective drugs uncovered repression of de novo lipogenesis as the convergent molecular pathway. We present FatTracer, a CRISPR screening platform to identify steatosis modulators and putative targets using APOB−/− and MTTP−/− organoids. From a screen targeting 35 genes implicated in lipid metabolism and/or NAFLD risk, FADS2 (fatty acid desaturase 2) emerged as an important determinant of hepatic steatosis. Enhancement of FADS2 expression increases polyunsaturated fatty acid abundancy which, in turn, reduces de novo lipogenesis. These organoid models facilitate study of steatosis etiology and drug targets

Investigazione del ruolo biologico di reelin, proteina extracellulare cerebrale, nelle cellule staminali neurali murine dell’adulto

Le cellule staminali neurali sono presenti, nel cervello dei mammiferi, in quelle aree dove la neurogenesi è mantenuta durante tutta la vita dell’organismo. Una caratteristica importante riguarda la loro abilità di proliferare e migrare nella materia cerebrale attratte dai siti di danno indotto, per esempio, da numerose malattie degenerative e dai tumori cerebrali. Il punto focale del mio dottorato è stato quello di studiare il ruolo di Reelin, proteina della matrice extracellulare fortemente implicata nello sviluppo cerebrale, nelle cellule staminali neurali murine. Mettendo a confronto le cellule “selvatiche” con quelle estratte da topi reeler, caratterizzate dalla mutazione negativa spontanea nel gene, ho osservato che l’assenza di Reelin rallenta la proliferazione delle cellule staminali neurali, così come la formazione delle neurosfere, aggregati sferici in sospensione tipici della crescita in vitro di queste cellule. Inoltre ho dimostrato un ruolo della proteina nel potenziale di differenziazione, favorendo la neuronogenesi in vitro senza modificarne la gliogenesi. Infine ho riscontrato l’incapacità delle cellule reeler di migrare nello stato aggregato chiamato “a catena”, la modalità di migrazione caratteristica delle cellule staminali neurali in vivo. Tutti questi effetti sono parzialmente recuperati dalla somministrazione della proteina esogena alle cellule reeler o con la diretta ingegnerizzazione per ripristinarne l’espressione endogena. Le conclusioni ricavate dal mio studio assegnano a Reelin un ruolo chiave nella biologia delle cellule staminali neurali adulte, intervenendo nella proliferazione, nel differenziamento e nella migrazione, le tre principali caratteristiche che guidano il processo di rigenerazione del tessuto danneggiato.In the adult mammalian brain, multipotential neural stem cells persist throughout life in those areas where neurogenesis is maintained. A distinctive trait of these cells is their ability to self-renew and to migrate through brain matter to sites of injury, such as those of occurrence of gliomas and neurodegenerative deseases. The aim of my doctorate study was the role of Reelin, an extracellular matrix protein deeply involved in brain development, in newborn mouse neural stem cells (NSCs). By comparing wild type and Reelin knock out reeler stem cells, I show that the absence of Reelin negatively affects proliferation and neurosphere-forming ability, as well as in vitro neuronal differentiation potential. Notably, reeler NSCs are not able to migrate in chains, a migration mode typical of neural precursors homing to injury sites in adult CNS. All these effects are partially rescued by ectopic Reelin supplementation. Overall, my results indicate that Reelin affects all three major features of post-natal NSCs, and that it is required for the proper homing of NSCs to injury sites in adult brain

Organoid models of fibrolamellar carcinoma mutations reveal hepatocyte transdifferentiation through cooperative BAP1 and PRKAR2A loss

Abstract Fibrolamellar carcinoma (FLC) is a lethal primary liver cancer, affecting young patients in absence of chronic liver disease. Molecular understanding of FLC tumorigenesis is limited, partly due to the scarcity of experimental models. Here, we CRISPR-engineer human hepatocyte organoids to recreate different FLC backgrounds, including the predominant genetic alteration, the DNAJB1-PRKACA fusion, as well as a recently reported background of FLC-like tumors, encompassing inactivating mutations of BAP1 and PRKAR2A. Phenotypic characterizations and comparisons with primary FLC tumor samples revealed mutant organoid-tumor similarities. All FLC mutations caused hepatocyte dedifferentiation, yet only combined loss of BAP1 and PRKAR2A resulted in hepatocyte transdifferentiation into liver ductal/progenitor-like cells that could exclusively grow in a ductal cell environment. BAP1-mutant hepatocytes represent primed cells attempting to proliferate in this cAMP-stimulating environment, but require concomitant PRKAR2A loss to overcome cell cycle arrest. In all analyses, DNAJB1-PRKACA fus organoids presented with milder phenotypes, suggesting differences between FLC genetic backgrounds, or for example the need for additional mutations, interactions with niche cells, or a different cell-of-origin. These engineered human organoid models facilitate the study of FLC