Researchers have developed a method to analyze extracellular vesicles (EVs) in blood as a potential early detection tool for Parkinson’s disease (PD). By isolating EVs and examining their contents, they identified elevated levels of phosphorylated α-synuclein in PD patients. If successful, this approach could enable noninvasive blood-based diagnostic methods for PD and other neurodegenerative diseases.
A new study led by researchers at Harvard University has developed a novel method to more accurately measure specific proteins in extracellular vesicles (EVs) present in plasma, focusing on α-synuclein, a protein linked to neurodegenerative diseases.
EVs are microscopic particles that all cells release and that transport a variety of materials, including proteins, within their membranes.
Due to the potential of EVs as biomarkers for diseases such as Parkinson's, the analysis of specific proteins contained in these vesicles has been a growing focus in biomedical research.
However, the low quantity of EVs in the blood and the difficulties in separating them from free proteins in plasma make it difficult to accurately assess the content of these vesicles.
The study aimed to develop and validate reliable methods to confirm whether proteins such as α-synuclein are indeed encapsulated within EVs, as well as to quantify the relative amount of these proteins within EVs compared to total plasma. α-synuclein, in its phosphorylated form (p-α-synuclein), has been associated with the development of diseases such as Parkinson's and dementia with Lewy bodies, where this variant of the protein is known to accumulate abnormally.
To overcome the challenges of isolating and measuring proteins inside EVs, the Harvard team used an integrated approach with several advanced technologies.
Size Exclusion Chromatography (SEC) was one of the techniques, this method has been optimized to efficiently isolate EVs by separating them based on their size. SEC allows for purer isolation of EVs and reduces contamination with free proteins in the plasma, a critical step for obtaining reliable results.
In addition, the Protease Protection assay uses a protease to degrade proteins that are outside the EVs. Because the lipid membrane of EVs protects their internal contents, the α-synuclein contained within them remains intact, while the free proteins in the plasma are broken down.
This method allows them to verify whether α-synuclein is actually encapsulated within EVs or just present in the plasma as a free protein.
Finally, they used Single Molecule Array Digital ELISAs (Simoa). This state-of-the-art technique is known for its ultrasensitive ability to detect proteins at minute levels.
Simoa digital ELISAs were employed to measure the levels of total α-synuclein and phosphorylated α-synuclein within EVs. This sensitivity is essential for analyzing proteins at very low concentrations, which are common in EVs.
The analysis of the data revealed crucial information about α-synuclein in EVs. It was confirmed that only a small fraction of the total α-synuclein in plasma is present inside EVs. This supports the idea that EVs carry the protein in detectable quantities, albeit in a small proportion.
The researchers observed that the ratio of phosphorylated α-synuclein (p-α-synuclein) to total α-synuclein was higher inside EVs than outside. This phosphorylated form of the protein is particularly relevant, as it is associated with the pathology of neurodegenerative diseases.
The enriched presence of p-α-synuclein inside EVs suggests that these vesicles may be selectively loaded with this protein variant, which could be an important indicator of processes related to the development of the disease.
Extending their analysis, the researchers applied the method to plasma samples from patients with Parkinson's and dementia with Lewy bodies.
By measuring the levels of total and phosphorylated α-synuclein in these samples, the team demonstrated that their protocol is effective in detecting EV-associated proteins in patients, which could open the door to using these techniques as diagnostic tools. This is especially relevant for neurodegenerative conditions, where α-synuclein plays a key role.
This study provides a new approach to assessing proteins within EVs, allowing scientists to distinguish between encapsulated proteins and free proteins in plasma.
With this advancement, it becomes possible to use EVs as biomarkers for neurodegenerative diseases, which could have a major impact on the early detection and monitoring of these conditions.
Future research should focus on refining these methods, as well as expanding them to other patient groups and exploring additional proteins contained in EVs that may be linked to other disorders.
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Measurement of α-synuclein as protein cargo in plasma extracellular vesicles
Tal Gilboa, Dmitry Ter-Ovanesyan, Shih-Chin Wang, and David R. Walt
PNAS. October 30, 2024. 121 (45) e2408949121
Abstract:
Extracellular vesicles (EVs) are released by all cells and hold great promise as a class of biomarkers. This promise has led to increased interest in measuring EV proteins from both total EVs as well as brain-derived EVs in plasma. However, measuring cargo proteins in EVs has been challenging because EVs are present at low levels, and EV isolation methods are imperfect at separating EVs from free proteins. Thus, knowing whether a protein measured after EV isolation is truly inside EVs is difficult. In this study, we developed methods to measure whether a protein is inside EVs and quantify the ratio of a protein in EVs relative to total plasma. To achieve this, we combined a high-yield size-exclusion chromatography protocol with an optimized protease protection assay and Single Molecule Array (Simoa) digital enzyme-linked immunoassays (ELISAs) for ultrasensitive measurement of proteins inside EVs. We applied these methods to analyze α-synuclein and confirmed that a small fraction of the total plasma α-synuclein is inside EVs. Additionally, we developed a highly sensitive Simoa assay for phosphorylated α-synuclein (phosphorylated at the Ser129 residue). We found enrichment in the phosphorylated α-synuclein to total α-synuclein ratio inside EVs relative to outside EVs. Finally, we applied the methods we developed to measure total and phosphorylated α-synuclein inside EVs from Parkinson’s disease and Lewy body dementia patient samples. This work provides a framework for determining the levels of proteins in EVs and represents an important step in the development of EV diagnostics for diseases of the brain, as well as other organs.
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