Adenosine-to-inosine (A-to-I) RNA editing is an essential epitranscriptomic modification that modulates gene expression in human transcriptomes (Picardi et al., 2015). Dysregulation of RNA editing has been associated with several diseases, including neurological and neurodegenerative disorders such as brain ischemia, schizophrenia, epilepsy, Alzheimer’s disease (AD), and cancer (Hosaka et al., 2021; Karagianni et al., 2022, 2024; Picardi et al., 2015). A potential link between aberrant RNA editing and amyotrophic lateral sclerosis (ALS) has also been proposed, but its precise role remains poorly understood. Understanding the involvement of RNA editing in ALS could uncover molecular mechanisms underlying the onset and progression of the disease, paving the way for novel diagnostic and therapeutic approaches. According to the "Japanese hypothesis", reduced editing at the Q/R site in GRIA2 mRNA mediated by ADAR2, coupled with decreased ADAR2 expression, should contribute to motor neuron death via AMPA receptor excitotoxicity in sporadic ALS (Yukio Kawahara*, 2004). Although supported by laser microdissected motor neurons from post-mortem tissues (Gregory et al., 2020; Yukio Kawahara*, 2004) and a conditional ADAR2 mouse model (Naito et al., 2023; Sasaki et al., 2014; Yamashita et al., 2013; Yamashita & Kwak, 2019), further evidence is required. To fill this gap, we conducted an extensive analysis of A-to-I RNA editing in two large ALS cohorts. First, we examined the TARGET ALS cohort (CHAPTER 1), comprising 1,373 RNA-sequencing datasets from post-mortem central nervous system tissues of individuals with sporadic ALS and controls. Second, we analyzed RNA-seq data from motor neurons derived from induced pluripotent stem cells (iPSCs) of 249 donors in the ANSWER ALS cohort (CHAPTER 2), including sporadic ALS patients and healthy controls. Notably, our findings indicate an alteration in the activity of RNA editing in ALS, mainly related to neuroinflammation. Since no cellular models are currently available to investigate the role of RNA editing in ALS, we developed an inducible motor neuron model using HUES 03 embryonic stem cells (hESCs) with downregulated ADAR2. Our cell model should allow the study of A-to-I editing dysfunction in ALS (CHAPTER 3) and provide a controlled system to validate the "Japanese hypothesis" and the physiological role of recoding RNA editing in neurodegeneration. During the PhD course, I spend five months at the Medical University of Vienna in Professor Michael F. Jantsch’s laboratory, where I studied the interplay between RNA modifications, focusing the attention on the relationship between m6A methylation and A-to-I RNA editing. Recent studies challenge the traditionally accepted antagonistic relationship between these two modifications (Xiang et al., 2018), suggesting instead that m6A might facilitate A-to-I editing by enhancing ADAR enzyme recruitment or activity. To explore this hypothesis (CHAPTER 4), I performed co-transfection experiments involving an RNA guide, a λN-ADAR2 deaminase domain (DD) fusion vector, and a Meg3 expression vector. This system allowed for site-specific A-to-I conversion and subsequent evaluation of how this editing influences m6A methylation at the targeted site. Although the high number of screened samples, our results suggest that further investigations are needed to assess the functional role of RNA editing in ALS and the relationship with other concurrent modifications. Long-read-based sequencing platforms could help in unveiling yet unknown aspects of RNA editing in ALS, enhancing our understanding of post-transcriptional gene regulation in neurodegenerative diseases.
L’editing dell’RNA da adenosina a inosina (A-to-I) rappresenta una modifica epitranscrittomica fondamentale nel modulare l’espressione genica nei trascrittomi umani. Studi precedenti hanno evidenziato come la disregolazione di questo processo sia associata a diverse malattie, tra cui patologie neurologiche e neurodegenerative come ischemia cerebrale, schizofrenia, epilessia, malattia di Alzheimer e cancro. Un possibile legame è stato ipotizzato anche tra editing aberrante dell’RNA e sclerosi laterale amiotrofica (SLA), sebbene il ruolo preciso di questa modifica nella SLA rimanga poco chiaro. Esaminare il coinvolgimento dell’editing dell’RNA nella SLA potrebbe offrire nuove prospettive sui meccanismi molecolari della malattia e su potenziali approcci terapeutici. La cosiddetta “ipotesi giapponese” propone che una riduzione dell’editing nel sito Q/R nell’mRNA di GRIA2, mediata dall’enzima ADAR2, contribuisca alla morte dei motoneuroni tramite l’eccitotossicità dei recettori AMPA. Questa riduzione è legata a una diminuzione dell’espressione di ADAR2. Le evidenze a supporto di questa ipotesi derivano da studi su motoneuroni microdissezionati da tessuti post-mortem di pazienti con SLA sporadica e da modelli murini condizionali privi di ADAR2. Tuttavia, per confermare e ampliare queste osservazioni, sono necessarie ulteriori indagini. A tale scopo, è stata condotta un’analisi approfondita dell’editing A-to-I dell’RNA su due grandi coorti di pazienti con SLA. La prima, la coorte TARGET ALS, include 1.373 dataset di sequenziamento dell’RNA ottenuti da tessuti del sistema nervoso centrale post-mortem di pazienti con SLA sporadica e controlli sani. La seconda, ANSWER ALS, comprende dati di RNA-seq derivati da motoneuroni generati da cellule staminali pluripotenti indotte (iPSC) di 249 donatori, inclusi pazienti con SLA e soggetti sani. I risultati ottenuti mostrano un’alterazione dell’attività di editing A-to-I nella SLA, principalmente associata alla neuroinfiammazione. Poiché non esistono modelli cellulari consolidati per studiare il ruolo dell’editing dell’RNA nella SLA, è stato sviluppato un modello innovativo di motoneuroni inducibili a partire da cellule staminali embrionali HUES 03 con ridotta espressione di ADAR2. Questo sistema sperimentale consente di analizzare specificamente la disfunzione dell’editing A-to-I nella SLA, validando ipotesi come quella giapponese e studiando il ruolo fisiologico di questa modifica nella neurodegenerazione. Nel contesto del mio dottorato, ho trascorso cinque mesi presso l’Università Medica di Vienna, sotto la supervisione del Professor Michael F. Jantsch, per indagare l’interazione tra le modifiche epitranscrittomiche. In particolare, mi sono concentrato sulla relazione tra metilazione m6A e editing A-to-I. Studi recenti suggeriscono che la m6A potrebbe facilitare l’editing A-to-I, favorendo il reclutamento o l’attività degli enzimi ADAR. Per approfondire questa ipotesi, ho sviluppato un sistema sperimentale basato sulla co-trasfezione di una guida RNA, un vettore λN-ADAR2 contenente il dominio deaminasi, e un vettore per l’espressione di Meg3. Questo modello ha permesso di valutare come l’editing A-to-I influenzi la metilazione m6A sullo stesso sito target. Nonostante il grande numero di campioni analizzati, ulteriori studi sono necessari per comprendere il ruolo funzionale dell’editing A-to-I nella SLA e la sua interazione con altre modifiche epitranscrittomiche. Tecnologie di sequenziamento a lettura lunga potrebbero rivelare nuovi dettagli di questo processo, migliorando la comprensione dei meccanismi di regolazione genica post-trascrizionale nelle malattie neurodegenerative.
Analisi dell'editing A-to-I dell'RNA nella sclerosi laterale amiotrofica
SPADAVECCHIA, PAOLA
2025
Abstract
Adenosine-to-inosine (A-to-I) RNA editing is an essential epitranscriptomic modification that modulates gene expression in human transcriptomes (Picardi et al., 2015). Dysregulation of RNA editing has been associated with several diseases, including neurological and neurodegenerative disorders such as brain ischemia, schizophrenia, epilepsy, Alzheimer’s disease (AD), and cancer (Hosaka et al., 2021; Karagianni et al., 2022, 2024; Picardi et al., 2015). A potential link between aberrant RNA editing and amyotrophic lateral sclerosis (ALS) has also been proposed, but its precise role remains poorly understood. Understanding the involvement of RNA editing in ALS could uncover molecular mechanisms underlying the onset and progression of the disease, paving the way for novel diagnostic and therapeutic approaches. According to the "Japanese hypothesis", reduced editing at the Q/R site in GRIA2 mRNA mediated by ADAR2, coupled with decreased ADAR2 expression, should contribute to motor neuron death via AMPA receptor excitotoxicity in sporadic ALS (Yukio Kawahara*, 2004). Although supported by laser microdissected motor neurons from post-mortem tissues (Gregory et al., 2020; Yukio Kawahara*, 2004) and a conditional ADAR2 mouse model (Naito et al., 2023; Sasaki et al., 2014; Yamashita et al., 2013; Yamashita & Kwak, 2019), further evidence is required. To fill this gap, we conducted an extensive analysis of A-to-I RNA editing in two large ALS cohorts. First, we examined the TARGET ALS cohort (CHAPTER 1), comprising 1,373 RNA-sequencing datasets from post-mortem central nervous system tissues of individuals with sporadic ALS and controls. Second, we analyzed RNA-seq data from motor neurons derived from induced pluripotent stem cells (iPSCs) of 249 donors in the ANSWER ALS cohort (CHAPTER 2), including sporadic ALS patients and healthy controls. Notably, our findings indicate an alteration in the activity of RNA editing in ALS, mainly related to neuroinflammation. Since no cellular models are currently available to investigate the role of RNA editing in ALS, we developed an inducible motor neuron model using HUES 03 embryonic stem cells (hESCs) with downregulated ADAR2. Our cell model should allow the study of A-to-I editing dysfunction in ALS (CHAPTER 3) and provide a controlled system to validate the "Japanese hypothesis" and the physiological role of recoding RNA editing in neurodegeneration. During the PhD course, I spend five months at the Medical University of Vienna in Professor Michael F. Jantsch’s laboratory, where I studied the interplay between RNA modifications, focusing the attention on the relationship between m6A methylation and A-to-I RNA editing. Recent studies challenge the traditionally accepted antagonistic relationship between these two modifications (Xiang et al., 2018), suggesting instead that m6A might facilitate A-to-I editing by enhancing ADAR enzyme recruitment or activity. To explore this hypothesis (CHAPTER 4), I performed co-transfection experiments involving an RNA guide, a λN-ADAR2 deaminase domain (DD) fusion vector, and a Meg3 expression vector. This system allowed for site-specific A-to-I conversion and subsequent evaluation of how this editing influences m6A methylation at the targeted site. Although the high number of screened samples, our results suggest that further investigations are needed to assess the functional role of RNA editing in ALS and the relationship with other concurrent modifications. Long-read-based sequencing platforms could help in unveiling yet unknown aspects of RNA editing in ALS, enhancing our understanding of post-transcriptional gene regulation in neurodegenerative diseases.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14242/217866
URN:NBN:IT:UNIBA-217866