Isik, Fatma Busra
(2025)
Plasma small extracellular vesicles as biomarkers in neurodegenerative dementias: focusing on microglial enrichment in dementia with Lewy bodies.
PhD thesis, University of Nottingham.
Abstract
There is an urgent clinical need for identifying blood-based biomarkers that can aid distinguishing between the two most common neurodegenerative dementias, AD and DLB. Advancing our understanding of its molecular pathogenesis is essential for identifying novel biomarkers and therapeutic targets for them. Extracellular vesicles (EV) are cell-derived vesicles with lipid bilayer membrane which carry biologically active proteins, lipids and nucleic acids including messenger RNA (mRNA) and small non-coding RNAs from their cells of origin. Because of their potential for transferring biological information, and ability for cell-to-cell communication, EV can be utilised as a tool to develop blood-based biomarkers for differentiating DLB from AD.
I investigated the importance of extracellular vesicles (EV) in understanding the pathology of DLB primarily through in silico analyses of EV RNA sequencing. I replicated an established protocol for enriching plasma EV of neuronal origin and adapted the protocol for isolating microglia origin enriched EV. I performed Illumina RNA sequencing analysis from EV enriched from neuronal, astrocyte, and microglial origins. I assessed differential expression of mRNA and non-coding RNAs between groups of AD, DLB and healthy control individuals. In chapter 5, I identified 4,278 potential target genes of differentially expressed miRNAs in individuals with DLB. Key enriched processes included transcriptional regulation, apoptosis, autophagy, and neuronal development, and enriched molecular functions, protein kinase and ubiquitin-protein transferase activity. In chapter 5, I identified 28 differentially expressed genes (DEG) including SLC6A18, QRICH2, ANOS1, ZBTB16 and MT-RNR2 with the sequencing analysis of plasma small extracellular vesicles (SEV) from people with AD. Subsequently, in chapter 6, the analysis of SEV from patients with AD revealed that tRNA-Glu-CTC-1-1 was upregulated in total plasma SEV, while mitochondrial tRNAs like MT-TV and MT-TE were downregulated in neuronal and astrocyte origin enriched. Additionally, 22 miRNAs, including hsa-miR-185-5p and hsa-miR-142-5p, were differentially expressed.
In chapter 7, I successfully developed a protocol for enriching microglial origin SEV using antibody against TMEM119, a cell-surface protein that is expressed in microglia. RNA sequencing analysis comparing microglial origin enriched SEV from plasma of people with DLB and AD indicated PRDX6, and TMEM97 were significantly downregulated in DLB. In contrast, the genes MIR28, TSPAN4, and SLC13A4 were found significantly upregulated in DLB. To conclude, I demonstrated the viability of conducting comprehensive RNA-Seq analysis using enriched SEV samples from plasma. This method allowed for the identification of potential blood-based diagnostic biomarkers that can aid differentiating between DLB and AD.
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