| Summary: | Astrocytes are glial cells within the brain responsible for maintaining healthy brain function. They also have the ability to react to harmful stimuli by changing into a reactive phenotype in order to protect the brain alongside microglia. Extracellular vesicles (EVs) are released from all cell types within the central nervous system (CNS) and are thought to represent the phenotype of the cell at the time of release. Therefore astrocyte-derived EVs (ADEVs) were hypothesised to change to represent the reactive state of the astrocytes, making them useful tools to monitor brain health. The aim of this thesis was to better understand ADEVs from both quiescent and reactive astrocytes, as well as how they may change in disease.
Characterisation of serum-free and serum-cultured human primary astrocytes was completed through morphological analysis as well as RNA- sequencing and mass spectrometry analysis using a multi-omics approach, which found large differences in gene expression despite few differences in protein expression. Serum-free characterisation identified a quiescent phenotype which contrasted with serum cultures which mimicked a reactive phenotype. ADEVs were then isolated using ultrafiltration of conditioned media followed by size exclusion chromatography
(SEC). Serum-cultured ADEVs were identified as smaller using nanoparticle tracking analysis (NTA) yet displayed a similar morphology to serum-free ADEVs. Mass spectrometry produced a detailed ADEV dataset for both models for future investigation. Tissue-derived EVs (BDEVs) were also isolated from frontal lobe tissue of Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS) patients as well as age and sex-matched controls to investigate how EVs are altered in disease. Mass spectrometry was completed on the BDEVs producing a second dataset that was compared to the ADEVs. Finally, ADEV functionality was explored by treating quiescent, serum-free astrocytes with reactive, serum-cultured ADEVs and exploring gene expression changes. ATP concentrations within cells were also explored to investigate whether EVs had the potential for glycolytic activity, however, glycolysis was not discovered during this work.
In conclusion, serum elicits a reactive response in quiescent, serum-free astrocytes leading to morphological and transcriptional changes. These changes are reflected in the astrocyte secretome and their ADEVs suggesting astrocyte health can be monitored using ADEVs. Protein datasets were produced for ADEVs and BDEVs for future comparisons creating a foundation for future ADEV and BDEV research using the models characterised in this work.
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