Alzheimer's disease (AD) and Frontotemporal Dementia (FTD) are complex neurodegenerative disorders that are often difficult to distinguish clinically due to the overlap of most of their symptoms. The currently available biomarkers, which are detected in biological fluids or through neuroimaging techniques, still have limitations in terms of diagnostic sensitivity and specificity. In this context, extracellular vesicles (EVs), particularly small EVs (SEVs), are emerging as a promising, non-invasive source of diagnostic biomarkers. These particles carry microRNAs (miRNAs), which are small non-coding RNAs that regulate gene expression at the post-transcriptional level and are often deregulated in neurodegenerative diseases. In this study, the miRNA cargo of SEVs derived from plasma samples of 20 AD patients, 13 FTD patients and 20 healthy controls (HC) was analysed with the aim of identifying miRNAs significantly deregulated in the two pathologies. The isolation of EVs was achieved by differential centrifugation, and their characterisation was carried out by performing nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). This approach allowed to enrich SEVs and separate them from large EVs (LEVs). MiRNA libraries were generated using the Small RNA-Seq Library Prep Kit (Lexogen). The samples were then sequenced on an Illumina NextSeq 500 platform and finally analysed through bioinformatics approaches. The results obtained revealed 339 differentially expressed (DE) miRNAs in FTD and 291 in AD compared to HC. Notably, 74 miRNAs were commonly deregulated in both diseases: 38 shared the same expression trend, whereas 36 showed an opposite trend. To identify potential biomarkers that can distinguish AD from FTD, 7 DE miRNAs, which exhibited a significantly high difference in their respective fold-changes, were selected among those shared by both conditions. Of these, 5 miRNAs showed opposite trends between AD and FTD, while 2 miRNAs shared the same expression patterns in both diseases. These findings highlight the diagnostic potential of EV-derived miRNAs and their importance in understanding the molecular mechanisms underlying dementia.
La malattia di Alzheimer (AD) e la demenza frontotemporale (FTD) sono due malattie neurodegenerative complesse che spesso presentano sovrapposizioni fenotipiche, rendendole difficili da distinguere clinicamente. Gli strumenti diagnostici attualmente disponibili mostrano ancora limitazioni in termini di sensibilità e specificità, ció ha generato un interesse significativo per l’analisi di nuovi biomarcatori, in particolare quelli contenuti nelle vescicole extracellulari (EV). Tra queste, le vescicole piú piccole, le cosiddette small EV (SEV), rappresentano un veicolo promettente di microRNA (miRNA), piccoli RNA non codificanti coinvolti nella regolazione dell'espressione genica, la cui espressione risulta spesso alterata nelle patologie neurodegenerative. Nel presente studio, sono stati analizzati i profili di espressione dei miRNA contenuti nelle SEV isolate da campioni plasmatici di 20 pazienti con AD, 13 con FTD e 20 soggetti sani, con l’obiettivo di identificare i miRNA significativamente deregolati nelle due demenze. L'isolamento delle SEV è stato ottenuto tramite ultracentrifugazione, mentre la loro caratterizzazione è stata effettuata mediante analisi di tracciamento delle nanoparticelle (NTA) e microscopia elettronica a trasmissione (TEM). I miRNA estratti da queste vescicole sono stati sequenziati mediante tecnologia NGS (Next Generation Sequencing) e successivamente sottoposti ad analisi bioinformatiche. L’analisi ha consentito l’identificazione di 339 miRNA differenzialmente espressi nei campioni FTD e 291 nei campioni AD rispetto ai controlli. In particolare, 74 miRNA risultavano deregolati in entrambe le patologie. Di questi, 38 miRNA manifestavano un profilo di espressione comune, mentre 36 mostravano un andamento opposto. Al fine di identificare potenziali biomarcatori in grado di distinguere le due patologie, 7 miRNA differenzialmente espressi sono stati selezionati, in virtù della significativa differenza nei profili di espressione osservata tra AD e FTD. Di questi, 5 miRNA hanno mostrato trend di espressione opposti tra le due malattie, mentre 2 miRNA hanno condiviso gli stessi pattern di espressione in entrambe le patologie. I dati ottenuti evidenziano che questi miRNA possono rappresentare futuri biomarcatori utili per la diagnosi differenziale, oltre a fornire nuove informazioni sui meccanismi molecolari alla base della neurodegenerazione.
Identificazione di RNA non codificanti deregolati nelle vescicole extracellulari di pazienti affetti da Alzheimer e demenza frontotemporale
BUSACCA, MARIA
2024/2025
Abstract
Alzheimer's disease (AD) and Frontotemporal Dementia (FTD) are complex neurodegenerative disorders that are often difficult to distinguish clinically due to the overlap of most of their symptoms. The currently available biomarkers, which are detected in biological fluids or through neuroimaging techniques, still have limitations in terms of diagnostic sensitivity and specificity. In this context, extracellular vesicles (EVs), particularly small EVs (SEVs), are emerging as a promising, non-invasive source of diagnostic biomarkers. These particles carry microRNAs (miRNAs), which are small non-coding RNAs that regulate gene expression at the post-transcriptional level and are often deregulated in neurodegenerative diseases. In this study, the miRNA cargo of SEVs derived from plasma samples of 20 AD patients, 13 FTD patients and 20 healthy controls (HC) was analysed with the aim of identifying miRNAs significantly deregulated in the two pathologies. The isolation of EVs was achieved by differential centrifugation, and their characterisation was carried out by performing nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). This approach allowed to enrich SEVs and separate them from large EVs (LEVs). MiRNA libraries were generated using the Small RNA-Seq Library Prep Kit (Lexogen). The samples were then sequenced on an Illumina NextSeq 500 platform and finally analysed through bioinformatics approaches. The results obtained revealed 339 differentially expressed (DE) miRNAs in FTD and 291 in AD compared to HC. Notably, 74 miRNAs were commonly deregulated in both diseases: 38 shared the same expression trend, whereas 36 showed an opposite trend. To identify potential biomarkers that can distinguish AD from FTD, 7 DE miRNAs, which exhibited a significantly high difference in their respective fold-changes, were selected among those shared by both conditions. Of these, 5 miRNAs showed opposite trends between AD and FTD, while 2 miRNAs shared the same expression patterns in both diseases. These findings highlight the diagnostic potential of EV-derived miRNAs and their importance in understanding the molecular mechanisms underlying dementia.| File | Dimensione | Formato | |
|---|---|---|---|
|
tesi.busacca.maria.pdf
non disponibili
Descrizione: Tesi Sperimentale
Dimensione
2.58 MB
Formato
Adobe PDF
|
2.58 MB | Adobe PDF | Richiedi una copia |
È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/30225