FUS affects circular RNA expression in murine embryonic stem cell-derived motor neurons

The RNA-binding protein FUS participates in several RNA biosynthetic processes and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Here we report that FUS controls back-splicing reactions leading to circular RNA (circRNA) production. We identified circRNAs expressed in in vitro -derived mouse motor neurons (MNs) and determined that the production of a considerable number of these circRNAs is regulated by FUS. Using RNAi and overexpression of wild-type and ALS-asso- ciated FUS mutants, we directly correlate the modulation of circRNA biogenesis with alteration of FUS nuclear levels and with putative toxic gain of function activities. We also demonstrate that FUS regulates circRNA biogenesis by binding the introns flanking the back-splicing junctions and that this control can be reproduced with artificial constructs. Most circRNAs are conserved in humans and specific ones are deregulated in human-induced pluripotent stem cell-derived MNs carrying the FUS P525L mutation associated with AL

Set up and optimization of a tandem mass spectrometry procedure for enzymatic assays of lysosomal storage disorders and feasibility evaluations for a newborn screening program

LSDs are a cluster of about 50 different inheritable pathologies mainly due to the deficit of lysosomal enzymes, which lead to severe and progressive multiorgan dysfunction. Biochemically well defined, they are characterized by an ubiquitous accumulation of undegraded macromolecules and, although individually rare, they present an overall incidence around 1 in 1500-7000 newborns. Main therapies for these pathologies are Enzyme Replacement and Hematopoietic Stem Cell Transplantation, both needing an early application to maximize their benefits; up to now, laboratory tests are carried out only following a particular clinical suspect and they allow a correct diagnosis when symptoms (sometimes irreversible) are already clearly developed. Other medical approaches, as enzyme enhancement therapy, substrate deprivation/reduction therapy and gene therapy, are on trial. Moreover, still very little is known on the individual determinants of therapeutic efficacy and, at the moment, the same therapeutic scheme is applied to all patients, although it is becoming clear that much better results could be obtained for each single patient if personalized therapeutic protocols could be defined. Both of these questions drive to the search of new diagnostic approaches able to detect patients in the population at the earliest possible time, in order to allow a very precocious application of therapies, and able to rapidly test enzymes bio-availability in blood, to facilitate the development of personalized therapeutic protocols, to reduce unnecessary treatments and side effects and to lead to a more cost-effective healthcare. Finally, recent studies on high number of samples, have reported an unexpected high incidence of these disorders, underlined how LSDs might have been since now underestimated. For all these reasons in the last years a new easy, cheap, and reliable diagnostic test, using Tandem Mass Spectrometry, was developed allowing to measure enzymatic activities in Dried Blood Spots (DBS). This work was addressed to the set up and optimization of this method for the detection of the enzymatic activities for six lysosomal storage disorders; the choice of the diseases to take into consideration in this study was made according to the availability of a therapy and of reagents for MS/MS analysis. The first part of the work was conducted at the Centogene Laboratory of Vienna; after the Gaucher and Niemann Pick A/B assays set up, they were multiplexed in a single test together with Pompe, Fabry and Krabbe assays. Then, the reliability and sensitivity of the method were investigated through a cross comparison work between two laboratories (the Vienna Centogene Laboratory and a research group of the Wadsworth Center of NY). An evaluation of the most critical steps of the protocol that needed to be optimized was made. Finally, a pilot newborn screening project was started in collaboration with the Hospital of Szeged. Ten thousand Hungarian anonymous samples were analyzed singly, then samples presenting low activities for one of the enzymes were re-tested in duplicate. Confirmed positive samples were sent to a third laboratory in Rostock for molecular analysis, so the detection of mutation for pathological samples and an evaluation of the false positive rate was possible. These studies confirm, not only the reliability of using DBS as biological specimens and the feasibility of the analysis by tandem mass spectrometry, but also the easy application of the method for screening intent. The second part of the work was carry out in Padova, in collaboration with the Tandem Mass Spectrometry Laboratory of the Department of Pediatrics. Here the multiplex assay was reproduced and the test for Mucopolysaccharidosis type I was added. Considering the preliminary results obtained in Vienna the protocol has been modified, by reducing the sample manipulation, through the introduction of an Ultra Performance Liquid Chromatography connected in line with the MS/MS spectrometer, and by optimizing the analytical parameters. The aim of the work was to maximize MS/MS sensitivity, precision and accuracy, to allow all enzyme activities to show an unambiguous difference between DBS obtained from healthy controls and LSD affected patients. The new defined protocol for enzyme activity detection described in this thesis will be potentially applied for several different purpose: it can be used both for the screening of wide populations, with the possibility to add new enzymatic tests for those disorders that will have a therapy available, or for the enzyme bioavailability determination in single patientsLe malattie da accumulo lisosomiale (LSD) rappresentano un gruppo di circa 50 disordini genetici, dovuti principalmente al deficit di specifiche idrolasi con conseguente accumulo di substrati non degradati nei lisosomi di organi e tessuti. Le LSD sono malattie molto complesse, gravemente debilitanti o letali, caratterizzate da un’estrema variabilità per quanto riguarda età di esordio, sintomatologia e rapidità di decorso clinico. Pur essendo singolarmente rare, esse presentano un’incidenza complessiva stimata di 1:1500-7000 nati vivi. Le principali terapie a disposizione, oltre alle cure palliative, sono la terapia enzimatica sostitutiva (ERT) e il trapianto di cellule staminali ematopoietiche; entrambe presentano massima efficacia se iniziate precocemente, prima cioè dell’insorgenza delle principali manifestazioni cliniche. Tuttavia, spesso una diagnosi precoce non è possibile poiché i test di laboratorio vengono eseguiti solo in seguito ad un evidente sospetto clinico. Altre strategie terapeutiche in fase di trial comprendono l’enzyme enhancement therapy, la terapia da inibizione/riduzione del substrato e la terapia genica. Ad oggi inoltre, i soggetti in trattamento vengono tutti sottoposti allo stesso protocollo terapeutico; sarebbe invece opportuno riuscire a sviluppare protocolli terapeutici personalizzati andando a valutare rapidamente la biodisponibilità dell’enzima somministrato, durante il follow up dei pazienti. Entrambe queste problematiche hanno portato, negli ultimi anni, alla ricerca di nuove tecniche diagnostiche, sia adatte per l’analisi di un elevato numero di campioni, che rispondenti alla necessità di permettere una veloce stima dell’attività degli enzimi di interesse in singoli soggetti. Il dosaggio enzimatico su Dried Blood Spot (DBS) mediante spettrometria in tandem massa si è rivelato un metodo ideale per la diagnosi delle malattie lisosomiali, poiché esso si distingue per affidabilità, sensibilità, rapidità e accuratezza e consente l’analisi enzimatica multipla in un elevato numero di campioni contemporaneamente. Infine, studi recenti di screening per alcune LSD condotti sia su ampie popolazioni sia su specifici gruppi a rischio (pazienti con infarto criptogenico o con insufficienza renale) hanno riportato un’incidenza complessiva sorprendentemente elevata, oltre a diversi casi, in particolare della malattia di Fabry, di mancato riconoscimento della patologia; ciò prova come questi disordini siano stati fino ad oggi sottovalutati, e sottolinea l’importanza di una corretta e tempestiva diagnosi. Questo lavoro è dedicato alla messa a punto e all’ottimizzazione di questa nuova metodica per lo studio dell’attività enzimatica per sei malattie da accumulo lisosomiale (Pompe, Fabry, Niemann Pick A/B, Gaucher, Krabbe e Mucopolisaccaridosi di tipo I). La scelta delle patologie è stata determinata sia, per ragioni etiche, dalla disponibilità di una terapia possibile, sia dalla disponibilità dei reagenti per la MSMS da parte del CDC di Atlanta. La prima parte del lavoro è stata svolta presso il laboratorio Centogene di Vienna, e ha previsto la messa a punto dei saggi per la malattia di Gaucher e Niemann Pick A/B, che sono stati successivamente unificati in un unico test insieme ai saggi per le malattie di Pompe, Fabry e Krabbe. In seguito, uno studio incrociato tra due laboratori (il laboratorio Centogene di Vienna e un gruppo di ricerca del Wadsworth Center di NY), su più di 400 campioni ha permesso di valutare l’affidabilità e la sensibilità della tecnica utilizzata, oltre ad individuare i passaggi critici del protocollo che sarebbero stati in seguito ottimizzati. Infine, nel corso di questo progetto è stato iniziato uno studio pilota di screening neonatale in collaborazione con l’Ospedale di Szeged (Ungheria). Diecimila campioni anonimi sono stati analizzati singolarmente; i campioni che presentavano una bassa attività per uno degli enzimi di interesse sono stati ri-analizzati in duplicato e nel caso di conferma, inviati ad un terzo laboratorio (Centogene di Rostock) per l’analisi molecolare. In questo modo è stata possibile sia un’identificazione delle mutazioni nei campioni patologici, sia una valutazione della percentuale di falsi positivi. Questi lavori hanno confermato non solo l’affidabilità nell’utilizzo dei DBS come campione biologico e la sensibilità della spettrometria in tandem massa per l’analisi, ma anche la facile organizzazione e applicazione di tale metodica per progetti di screening. La seconda parte del progetto è stata svolta a Padova in collaborazione con la sezione di spettrometria di massa del Dipartimento di Pediatria. Qui è stato riprodotto il saggio multiplo per i 5 enzimi sopra citati ed è stato aggiunto inoltre il test per la Mucopolisaccaridosi di tipo I. Tenendo conto dei risultati preliminari ottenuti a Vienna il protocollo è stato così modificato: è stata ridotta al minimo la parte preparativa dei campioni, sostituita da una cromatografia liquida collegata online con lo spettrometro di massa; inoltre, tutti i parametri analitici sono stati minuziosamente ottimizzati. Lo scopo è stato quello si massimizzare la sensibilità e precisione di rilevamento degli analiti per garantire una inconfutabile discriminazione tra soggetti sani e patologici. Questa nuova metodica può essere applicata per scopi diversi: sia per lo screening di ampie popolazioni, con la possibilità di aggiungere nuovi test al pannello delle malattie screenate via via che sarà per esse disponibile una terapia, sia per una rapida valutazione della bio-disponibilità dell’enzima in singoli soggett

Linc-NeD125 establishes a ceRNA network in Group 4 Medulloblastoma

Long noncoding RNAs (lncRNAs) are regarded as crucial regulators of cellular processes in Eukaryotes. About 40% of currently characterized lncRNAs are specifically expressed in central nervous system, where they are involved in critical neural functions. Consistently, lncRNA aberrant expression is associated to neurological disorders. We recently identified a novel human lncRNA, linc-NeD125, that is induced in response to neuronal differentiation stimulus both in tumour cell lines and in embryonic stem cells. Notably, linc-Ned125 is significantly upregulated in a specific and still largely uncharacterized subgroup (Group 4) of Medulloblastoma (MB), the most common malignant paediatric brain tumour. Combining mechanistic and functional studies, we unveiled a novel lncRNA-mediated miRNA sponge regulatory network, in which the cross-talk among linc-NeD125, microRNAs and four Group 4 MB driver gene transcripts may significantly contribute to Group 4 MB cancerogenesis

Targeting Brain Disease in MPSII: Preclinical Evaluation of IDS-Loaded PLGA Nanoparticles

Mucopolysaccharidosis type II (MPSII) is a lysosomal storage disorder due to the deficit of the enzyme iduronate 2-sulfatase (IDS), which leads to the accumulation of glycosaminoglycans in most organ-systems, including the brain, and resulting in neurological involvement in about two-thirds of the patients. The main treatment is represented by a weekly infusion of the functional enzyme, which cannot cross the blood-brain barrier and reach the central nervous system. In this study, a tailored nanomedicine approach based on brain-targeted polymeric nanoparticles (g7-NPs), loaded with the therapeutic enzyme, was exploited. Fibroblasts from MPSII patients were treated for 7 days with NPs loaded with the IDS enzyme; an induced IDS activity like the one detected in healthy cells was measured, together with a reduction of GAG content to non-pathological levels. An in vivo short-term study in MPSII mice was performed by weekly administration of g7-NPs-IDS. Biochemical, histological, and immunohistochemical evaluations of liver and brain were performed. The 6-weeks treatment produced a significant reduction of GAG deposits in liver and brain tissues, as well as a reduction of some neurological and inflammatory markers (i.e., LAMP2, CD68, GFAP), highlighting a general improvement of the brain pathology. The g7-NPs-IDS approach allowed a brain-targeted enzyme replacement therapy. Based on these positive results, the future aim will be to optimize NP formulation further to gain a higher efficacy of the proposed approach