Summary: Researchers have developed a method to analyze extracellular vesicles ( EVs ) in blood for early detection of Parkinson’s disease ( PD). The group identified a protein called activated -synuclein, which is present in increased levels in PD patients by isolating Vehicles and assessing their material.
Because these proteins changes can be detected before clinical signs and symptoms appear, this discovery might lead to earlier treatment. The method employs an ultra-sensible assay that can identify disease markers between plasma and completely EVs.
If powerful, this technique may enable non-invasive, blood-based tests for PD and another neurodegenerative disorders. If the check may effectively distinguish PD from various conditions, ongoing work will be done.
Important Information:
- EVs with raised oxidized levels of PD are related to this.
- External vesicles protect proteins biomarkers, helping maintain disease indicators.
- This blood-based medical approach may help earlier, non-invasive PD recognition.
Origin: Wyss Institute
Brain disorders like Parkinson’s ( PD ) or Alzheimer’s Disease ( AD ) start to develop in patients much earlier than when their first clinical symptoms appear.
There is currently no way to identify brain diseases at those pre-symptomatic periods, but treating patients at these early stages may slow down or even quit their condition.
So far, the particular head lesions caused by PD, for instance, can only be detected by analyzing brain samples, which can only be obtained subsequently.  ,
Scientists have been pursuing the new idea of “liquid samples,” which involves the simple recovery of body or other body fluids using non-invasive methods and their analysis for molecules originating from head and other good tissues in order to overcome this crucial barrier.
A particularly promising target in body fluids are “extracellular vesicles” ( EVs ), tiny membrane-bound sacs released by brain and other cells into their surrounding fluids.
These sacs may also have protected markers for the first beginnings of Parkinson’s and other mental conditions because they contain a wide range of molecules that can be specific to the types of cells that produce them, such as the mind.  ,
Despite recent advancements, EV experts have n’t been able to address the issue of whether particular biomarker molecules found in isolated EVs are strictly internalized or not specifically bound to their surface.
They are currently unable to draw clear opinions about cargo particles in EVs from all different types of tissues due to this difficulty.
Then, a creative staff led by , David Walt, Ph. D. at the Wyss Institute at Harvard University and , Brigham and Women’s Hospital , ( BWH) in Boston has solved this problem by adding a crucial step to an already validated , ultra-sensitive protocol.
By organically digesting all surface-bound enzymes from a purified EV people, they were able to specifically focus on goods inside of Vehicles while avoiding unspecified” contaminations.”
For the first time, they were able to accurately determine the small percentage of any proteins contained within EVs compared to how much of it was manifest free in full body blood using their improved protocol to determine the PD biomarker , -synuclein in blood.  ,
Importantly, they integrated this advancement with a recently developed ultra-sensitive detection assay for a form of phosphorylated RNA that increases as a result of PD and the concomitant condition Lewy Body Dementia.
Analyzing a cohort of patient samples, they could detect an enrichment of the pathological , ⍺-synuclein protein inside EVs relative to total plasma. The findings are published in , PNAS.
Over the past few decades, research on EVs in our and other groups has steadily improved our understanding of their complex biology and molecular composition.
However, validating and quantifying their true contents with precise measurements still presents formidable technical challenges, according to Wyss Core Faculty member Walt. The isolation of pure tissue-specific EVs from body fluids like blood or the cerebrospinal fluid surrounding the central nervous system, including the brain, and the isolation of body fluids like blood or the cerebrospinal fluid still poses serious technical challenges.
Our most recent work is providing a solution to help us get EVs free from contamination in order to use them as valuable sources for clinical biomarkers, as we demonstrate with the example of phosphorylated , -synuclein.
Walt, who is the faculty lead of the Wyss Institute’s Diagnostic Accelerator, is also the Hansjörg Wyss Professor of Biologically Inspired Engineering at Harvard Medical School ( HMS ), Professor of Pathology at Brigham and Women’s Hospital, and a Howard Hughes Medical Institute Professor.
From blood to EVs to biomarkers to diagnosis
The Walt group has been systematically putting crucial pieces into this technical jigsaw puzzle, especially motivated by the diagnostic potential of EVs for the early diagnosis of PD, AD, and other brain disorders.
With philanthropic support from , Good Ventures, the , Chan Zuckerberg Initiative, and more recently the , Michael J. Fox Foundation, they previously developed a , technical framework , for quantifying EVs and used this quantification to compare EV isolation methods from body fluids.
Their method combines a size exclusion chromatography ( SEC ) separation technique with ultra-sensible” Simoa assays to count single protein molecules associated with EVs that they captured and visualized with specific antibodies to recover the majority of EVs from biofluids.
By now, the team has engineered Simoa assays for a variety of EV-specific biomarkers and, importantly, excluded a widely used candidate surface protein, L1CAM, as a target to isolate brain-specific EVs, which provided the field with an important , course correction.
We used SEC isolation methods that we previously developed to isolate most EVs from plasma together with an optimized’proteinase protection assay’ to gently but effectively chew all proteins off the surface of isolated EVs while leaving the membrane-enclosed EV interior intact. This is a question that is conceptually simple but technically challenging. ” said co-first author , Dima Ter-Ovanesyan, Ph. D., who is a Senior Scientist at the Wyss Institute and spearheads the EV project with co-first author and Postdoctoral Fellow , Tal Gilboa, Ph. D.
Also, to measure , ⍺-synuclein at very low levels, Gilboa, together with Postdoctoral Fellow , Gina Wang, Ph. D. and Wyss Research Assistant , Sara Whiteman , in the Walt lab, developed a Simoa assay for , ⍺-synuclein that is much more sensitive that any previously reported assay.
Using this assay in their protocol, the team was able to determine that most of the , ⍺-synuclein in EVs isolated using their SEC protocol was protected and that this amount presented less than 5 % of total blood plasma , ⍺-synuclein.
Understanding this level is crucial for the eventual determination of measuring neuron-derived EVs because EVs that originate from a particular tissue, such as the brain, are expected to be uncommon in comparison to EVs from blood cells, where -synuclein is also expressed.  ,
Importantly, in addition to their ultra-sensitive Simoa assay that enabled them to detect the normal unmodified , ⍺-synuclein protein, they also developed an assay that is able to detect , ⍺-synuclein that becomes phosphorylated at a specific site ( pSer129 ) in the course of PD progression.
When we used our advanced method to analyze a cohort of blood samples from PD and Lewy Body Dementia patients as well as healthy control donors, we discovered that the ratio of phosphorylated, -synuclein to total, and -synuclein was two to threefold higher inside EVs than outside of EVs,” said Gilboa.
This was very exciting because it suggested that EVs might help prevent circulating phosphatases from phosphorylating proteins, which would otherwise render this highly informative mark.
The team is now looking into whether these tests can be used to distinguish PD patients from those who do n’t have the disease.  ,  ,
The work of David Walt’s team creates a technological tour-de-force that brings us closer and closer to a next-generation diagnostic platform with extraordinary potential. At this point, we are not far from using these extremely rich and telling cell-derived vesicles as a window to peak into the brains of patients without requiring surgery,” said Wyss Founding Director , Donald Ingber,  , M. D., Ph. D., who is also the , Judah Folkman Professor of Vascular Biology , at HMS and Boston Children’s Hospital and the , Hansjörg Wyss Professor of Biologically Inspired Engineering , at Harvard’s John A. Paulson School of Engineering and Applied Sciences.
Additional authors of the paper are George Church, Ph. D., a Wyss Core Faculty member and the Robert Winthrop Professor of Genetics at HMS and Alice Chen-Plotkin, M. D., the Parker Family Professor of Neurology at the Perelman School of Medicine at the University of Pennsylvania, Philadelphia, who have both collaborated with Walt’s group since the inception of the EV project, as well as George Kannarkat.
Funding: The work received grants from the Michael J. Grants. Fox Foundation ( Grant# 2021A017224 ), Chan Zuckerberg Initiative NeuroDegeneration Challenge Network, and Good Ventures. The Weizmann Institute of Science Women’s Postdoctoral Career Development Award was presented to Gilboa.
About this Parkinson’s disease research news
Author: Benjamin Boettner
Source: Wyss Institute
Contact: Benjamin Boettner – Wyss Institute
Image: The image is credited to Neuroscience News
Original Research: Closed access.
Measurement of -synuclein as a protein cargo in plasma extracellular vesicles by David Walt et al. PNAS
Abstract
In plasma extracellular vesicles, the cytokine molecule -synuclein is measured as a protein cargo.
All cells release extracellular vesicles ( EVs ), which have a lot of promise as a class of biomarkers. This promise has fueled more research into measuring EV proteins in plasma from both total and brain-derived EVs.
However, EVs ‘ ability to detect cargo proteins in them has been difficult due to the low levels of EVs present at low levels and poor EV isolation techniques for separating EVs from free proteins. Therefore, it’s challenging to determine whether a protein identified after EV isolation actually exists inside EVs.
In this study, we developed methods to determine whether a protein is inside EVs and determine how much protein is present in EVs in relation to total plasma.
For ultra-sensitive protein measurements inside EVs, we combined a high-yield size-exclusion chromatography protocol with a highly sensitive protease protection assay and Single Molecule Array ( Simoa ) digital enzyme-linked immunoassays ( ELISAs ) with an optimized protease protection assay.
We tested these techniques on EVs to determine whether only a small portion of the total plasma -synuclein is contained within EVs. Additionally, we developed a highly sensitive Simoa assay for phosphorylated α-synuclein ( phosphorylated at the Ser129 residue ).
In comparison to outside EVs, the phosphorylated -synuclein to total -synuclein ratio was found to be higher inside.
Finally, we compared the methods we developed to measure total and phosphorylated EVs from Parkinson’s disease and patient samples from Lewy Body Degeneration.
This work provides a framework for determining the protein levels in EVs and is a significant step in the development of EV diagnostics for conditions of the brain and other organs.
