Utilising exosomes for biomarkers of Alzheimer's disease

• Main Author: Thomas, Rhodri
• Published: Cardiff University 2017
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.738352

Exosomes are small, nanometre-sized, vesicles secreted by cells into the extracellular milieu. They have shown good potential for biomarkers of disease as they are peripherally available, and therefore non-invasively obtained, and are representative of the source cell. Discovering novel biomarkers of Alzheimer’s disease is a desperate need that exosomes may address. This thesis explored the utility of RNA within exosomes for biomarkers of Alzheimer’s disease. Initially exosomes were isolated from H4 (neuroglioma) and IMR-32 (neuroblastoma) cell-lines in culture. The exosomes were thoroughly characterised and the cell-lines used to establish bulk stocks of neural-derived exosomes for downstream assay development and analyses. As a pre-requisite of capturing neural-derived exosomes directly from biological fluids, a number of protein ligands were tested for affinity isolation of cell culture-derived exosomes. A working assay could not be developed so the direction of this thesis changed to a systems-wide approach. A large RNA sequencing dataset was produced from RNA derived from H4 cells and exosomes. By performing whole transcriptome sequencing, novel insights into exosomal-RNA were made. It was determined that the profile of RNA within exosomes is distinct from the source cell and enriched for species that suggested that RNA was actively sorted into these vesicles. A method was then developed for isolating exosomal-RNA from small volumes of plasma and measuring gene expression for multiple targets by quantitative polymerase chain reaction. This method was validated by showing sensitivity to small changes in exosome dose and able to detect brain-enriched gene targets. In conclusion, RNA appears to be non-randomly sorted into exosomes and thus sensitive to the state of the source cell. A method has been developed, and validated, for isolating exosomal-RNA from small volumes of plasma. This could prove of great use in the future for discovering novel, peripherally available, biomarkers of Alzheimer’s disease.