The role of RNA in synaptic physiology and neurodegeneration in synucleinopathies
Doctoral thesis
Date of Examination:2025-01-10
Date of issue:2025-01-23
Advisor:Prof. Dr. Tiago Fleming Outeiro
Referee:Prof. Dr. Tiago Fleming Outeiro
Referee:Prof. Dr. André Fischer
Referee:Prof. Dr. Silvio Rizzoli
Referee:Prof. Dr. Ralf Heinrich
Referee:Prof. Dr. Christine Stadelmann-Nessler
Referee:Prof. Dr. Christian Griesinger
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Description:Doctoral Dissertation Avika Chopra
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Abstract
English
Synucleinopathies are neurodegenerative disorders characterised by the pathological accumulation and aggregation of the protein alpha-synuclein (aSyn). Parkinson’s disease (PD), Dementia with Lewy bodies (DLB), multiple system atrophy (MSA), and pure autonomic failure (PAF) are among the widely known and studied synucleinopathies. Among these, PD is the most prevalent, affecting about 1% of the world’s population older than 60. PD can be classified into sporadic (sPD) and familial forms, with most cases being sporadic due to environmental risk factors including pesticide exposure, trauma, and chemical exposure. Familial PD, on the other hand, accounts for only 10% of the total cases. Clinically, PD is defined by motor symptoms such as bradykinesia, rigidity, resting tremors, and gait irregularities. It is now accepted that PD progression begins decades before an individual presents with these motor symptoms. This phase is known as the pre-motor phase and it can last for several years. It is marked by symptoms including REM sleep behaviour disorder (RBD), constipation, hyposmia, and depression. Currently, PD diagnosis relies on detecting the loss of dopaminergic neurons in substantia nigra Pars Compacta (SNpc) and the detection of aSyn aggregates in the brain, often confirmed post-mortem. At the onset of motor symptoms, due to the loss of more than 50% of dopaminergic neurons in SNpc, symptomatic intervention is the last resort. Hence, there is a pressing need for early diagnosing methods of PD enabling early intervention. Firstly, we attempted to identify possible RNA-based biomarkers in a transgenic mouse model expressing human A30P aSyn (aSyn Tg). We compared exosomes from aSyn Tg primary astrocytes and neurons with wild type (Wt). Our analysis revealed four significantly upregulated miRNAs when exosomes from aSyn Tg neurons were compared with Wt – mmu-miR-185, mmu-miR-137, mmu-miR-130a, and mmu-miR-324. Interestingly, no significant differences were observed in astrocytic exosomes. Target prediction of these miRNAs revealed genes associated with epigenetic regulations, mainly methylation, prompting further investigation in the role of RNA methylation in PD. Similarly, for our second aim, we examined isolated exosomes from human serum samples from individuals with PD and MSA and compared them with age-matched healthy controls (HCs). Our analysis revealed that 20 miRNAs were upregulated and 21 were downregulated in PD while in MSA, 30 miRNAs were upregulated and 28 were downregulated. Among the total up and downregulated miRNAs, for further analysis, we selected approximately ten from each list of miRNAs from PD and MSA vs. HCs. These miRNAs were chosen based on their expression levels and relevance based on previously published studies. Target gene prediction for the selected miRNAs in PD samples show that these genes were relevant in cancer, cell stress, autophagy, and infections. Similarly, in MSA, the target genes of the dysregulated miRNAs were largely linked to cellular inflammation, hypoxia, and cell death. In addition to miRNAs, epitranscriptomics is also an upcoming research branch for the identification of potential biomarkers. Among the several modifications, m6A methylation is one of the most prominently studied modifications. An increase in methylation has been correlated with decreased transcriptional and translational efficiency. Therefore, our third aim involved examining m6A RNA methylation as a potential biomarker, specifically to understand the role of methylation in ageing and neurodegeneration. We used aSyn Tg mouse as the model of pathology, and 3 mo (young) mice were compared with 15 mo (old) mice. Through m6A RNA immunoprecipitation sequencing (meRIP-seq), we observed hypermethylation of synaptic genes in 3 mo aSyn Tg vs. Wt mice. This methylation decreased with ageing, with synaptic genes becoming increasingly hypomethylated in 15 mo aSyn Tg vs. Wt mice. To further characterise the m6A landscape, we analysed the key m6A regulatory enzymes – including the writer, N6-Adenosine-Methyltransferase Complex Catalytic Subunit (METTL3); reader, YTH N6-methyladenosine RNA-binding protein (YTHDF1); and eraser, fat mass and obesity-associated protein (FTO) proteins using immunofluorescence imaging and immunoblotting. These proteins were present in different brain regions (cortex, striatum, hippocampus, and cerebellum) in Wt and aSyn Tg mice, and primary cortical neuronal cultures, however, their levels did not vary between pathological and healthy conditions. Interestingly, our findings indicated that METTL3 was not only located in the nucleus but also in the post-synaptic compartment in neuronal cultures. These findings suggest that alterations in the regulation of m6A RNA methylation may be associated with neurodegeneration and ageing and this level of epitranscriptomic regulation might play a significant role at the synapse. Overall, our research highlights the potential of miRNA profiles in exosomes as minimally invasive biomarkers. Due to their abundance in bodily fluids, we aimed to discover biomarkers that offer promise in the diagnosis of PD in the pre-motor phase and potentially identify individuals at risk of developing the disease later in life. This might provide significant potential as a first-line diagnostic biomarker. Exosomal miRNA retrieval showed potential as a diagnostic tool, while m6A methylation patterns might enable clinicians to identify individuals at a higher risk of developing PD. Further validation of consistent miRNAs and m6A methylation levels could contribute to a novel class of biomarkers, facilitating early detection and therapeutic strategies for PD and related synucleinopathies.
Keywords: Parkinson’s disease, Multiple system atrophy, miRNAs, exosomes, epitranscriptomics, m6A