Differential expression of proteins involved in sEV biogenesis and secretion in Parkinson’s disease and Dementia with Lewy bodies

Abstract

Exosomes, small extracellular vesicles (sEVs), play a pivotal role in cell-to-cell communication, carrying diverse biomolecules such as proteins, lipids, and nucleic acids. Their ability to transport these molecules makes them fundamental to various physiological processes and valuable in diagnosing and treating disorders of the central nervous system (CNS). To fully utilise sEVs potential, more attention is needed to understand not only the cargo, but also proteins and pathways involved in the biogenesis of EVs, particularly in the context of diseases. This thesis explores changes in sEV-associated proteins in synucleinopathies, notably Parkinson’s disease (PD) and dementia with Lewy bodies (DLB).Aims1. In this study I aim to measure levels of key sEV proteins by focusing on disease-specific brain regions such as the substantia nigra and hippocampus of patients with PD and DLB compared to healthy control individuals.2. Given that the sEV proteins in the endosomal pathway are also involved in the autophagy-lysosomal pathway (ALP), I also aim to understand whether changes in sEV proteins have an influence on the ALP in PD and DLB.3. Finally, I aim to replicate these findings from human brain samples in an in vitro cell model of PD using the SY5Y cell model by generating wild-type and mutant variants (E46K and A53T) of alpha-synuclein. Here I also aim to understand whether the wild-type or mutant variants of alpha-synuclein affect the ALP.MethodsThe primary method used in this study was the use of frozen human brain tissue to measure changes in key sEV and ALP proteins in disease. Formalin-fixed, paraffin-embedded (FFPE) tissue sections were also used to try and identify cell specific changes in key sEV proteins, particularly Alix. The SY5Y cell model was also used as a relevant in vitro model for Parkinson’s disease. Plasmid vector delivery was used to generate wild-type alpha-synuclein and familial point mutations of PD including the A53T-synuclein and E46K-synuclein mutation. The in vitro results were supported by additional data generated at the University of Oxford where a monoclonal alpha-synuclein knockout SY5Y cell was used as a control and either wild-type of mutant (E46K or A53T) alpha-synuclein was overexpressed in these cells via lentiviral transduction.OutcomesBy focusing on disease-specific brain regions such as the substantia nigra and hippocampus, significant disease and region-specific changes in proteins that are involved in sEV biogenesis and cargo sorting were identified. This included a significant decrease in levels of Alix in the substantia nigra of patients with PD, and the hippocampus for both patients with PD and DLB. Levels of TSG101 were also significantly decreased in the substantia nigra of PD patient samples compared to the healthy control group. Given the key roles of Alix and TSG101 in sEV biogenesis, cargo sorting into sEVs, and maintenance of the ALP, and endolysosomal trafficking, I next focused on the levels of ALP proteins in PD and DLB. I have explored how these changes in key sEV proteins interface with the endosomal system and the ALP, revealing their dual role in sEV formation and the degradation of unnecessary cellular proteins. Contrary to expectations, no significant alterations in the ALP were observed in PD and DLB patient samples compared to healthy controls, including key proteins such as P62, Atg7, Rab7 and LAMP2. This suggests that although sEV proteins are affected, this may not necessarily influence degradation pathways such as the ALP in the model presented in this thesis. One striking change observed in this thesis was a significant reduction in the ALP protein Beclin-1 in both PD and DLB, suggesting a possible interplay between sEVs and the induction of autophagy that may vary across different conditions. However, the in vitro cell model revealed no changes in key sEV and ALP proteins in wild-type overexpressing and mutant cell variants. Future studies will need to establish and validate this model further by inducing large aggregates using pre-formed fibrils, as these aggregates were not observed in the cell model presented here.The supporting results from the in vitro cell model from the University of Oxford also revealed no changes in key sEV proteins and in Beclin-1 when comparing alpha-synuclein knockout non-transduced control cells to wild-type overexpressing and mutant cell variants. This further emphasises the need for potentially more relevant disease models that can represent the pathology observed in human patient samples.The findings underscore the critical role of sEV-associated proteins in neurodegenerative diseases and highlight the necessity for advanced model systems, such as induced pluripotent stem cells (iPSCs), that more accurately reflect the disease state. This study not only contributes to a deeper understanding of sEVs in synucleinopathies but also emphasises the ongoing need for comprehensive research in this domain to unlock the full diagnostic and therapeutic potential of sEVs in CNS disorders.LimitationsLimitations of this study include a low number of human patient samples in disease and control groups. Large samples sizes for human patient studies are often required due to the heterogeneity of data obtained, and future work will need a larger cohort of patients. The primary method used for human patient sample analysis was western immunoblotting. This study will need to be followed up by more complementary approaches including immunohistochemistry and quantitative polymerase chain reaction to verify the findings and strengthen the data. Moreover, although alpha-synuclein was overexpressed in the wild-type and mutant models in SY5Y cells, a lack of large aggregates may have contributed to the lack of any changes in key sEV or ALP proteins. Future studies would require the use of exogenously added pre-formed fibrils to induce aggregation pathology that is observed in patients with PD and DLB.

Awarding Institution(s)

University of Plymouth

Supervisor

Konstantin Glebov, Robert Fern

Document Type

Thesis

Publication Date

2025

Embargo Period

2026-08-12

Deposit Date

August 2025

This document is currently not available here.

This item is under embargo until 12 August 2026

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