summary: Researchers have developed a method to analyze extracellular vesicles (EVs) in the blood for early detection of Parkinson’s disease (PD). By isolating EVs and assessing their contents, the researchers identified a protein called phosphorylated alpha-synuclein that appears at high levels in PD patients.
Because changes in these proteins can be detected before clinical symptoms appear, this discovery may allow for earlier diagnosis. This approach uses an ultrasensitive assay that can distinguish between disease markers in EVs and those not present in plasma.
If successful, this technology could enable non-invasive, blood-based diagnosis of PD and other neurodegenerative diseases. Ongoing research will determine whether this test can reliably distinguish PD from other conditions.
Important facts:
- Elevated levels of phosphorylated α-synuclein in EVs correlate with PD progression.
- Extracellular vesicles protect protein biomarkers and help preserve disease indicators.
- This blood-based diagnostic technique may enable earlier non-invasive PD detection.
sauce: Wyss Institute
Brain disorders such as Parkinson’s disease (PD) and Alzheimer’s disease (AD) begin to develop in patients much earlier than the first clinical symptoms appear.
Treating patients at these early stages could potentially slow or stop the progression of the disease, but there is currently no way to diagnose brain disease at this pre-symptomatic stage.
So far, certain brain lesions caused by PD, for example, can only be detected by analyzing brain biopsies, which are only available post-mortem.
To overcome this critical bottleneck, researchers have pursued a new concept called “liquid biopsy.” This involves using non-invasive procedures to easily extract blood and other body fluids and analyze molecules derived from the brain and other solid tissues.
A particularly promising target in body fluids are ‘extracellular vesicles’ (EVs), small membrane-enclosed sacs released by brain and other cells into surrounding body fluids.
These sacs contain a variety of molecules that are specific to the cell type that produces them, such as the brain, and therefore also hold protective biomarkers for the early onset of Parkinson’s disease and other brain diseases. Possibly.
However, despite recent advances, EV experts are still unsure whether the specific biomarker molecules measured on isolated EVs are strictly contained inside the EV or nonspecifically on the surface of the EV. It has not been possible to address the question of whether they are connected.
This challenge actually prevents us from making clear conclusions about cargo molecules from all types of tissues within EVs.
A collaborative team has now been formed, led by Dr. David Walt. The Wyss Institute at Harvard University and Brigham and Women’s Hospital (BWH) in Boston solved this problem by adding a key step to an already validated ultra-sensitive protocol.
By enzymatically digesting all surface-bound proteins from purified EV populations, we specifically target cargo that is protected inside EVs while eliminating non-specific “contamination” is completed.
By measuring the PD biomarker ⍺-synuclein in blood using an improved protocol, we accurately determine the small fraction of proteins contained within EVs and the amount of protein present free in total plasma. For the first time, I was able to make a decision.
Importantly, they integrated this advance with a newly developed ultrasensitive assay for the detection of a type of ⍺-synuclein that becomes increasingly phosphorylated during the progression of PD and the related disease Lewy body dementia.
When analyzing a cohort of patient samples, we were able to detect an enrichment of pathological ⍺-synuclein protein in EVs compared to total plasma. The survey results are PNAS.
“Study of EVs by our and other groups over the past several decades has steadily advanced our understanding of their complex biology and molecular composition.
“However, isolating pure tissue-specific EVs from body fluids such as blood and cerebrospinal fluid, which surrounds the central nervous system, including the brain, and verifying and quantifying their true contents through precise measurements remains intimidating. “There are technical challenges that need to be addressed,” Wyss Core said. Mr. Walt, a teacher.
“Our recent research provides a solution that helps fill this technological gap by phosphorylating ⍺-synuclein.”
Mr. Walt is Dean of the Diagnostic Accelerator at the Wyss Institute, the Hansjorg Wyss Professor of Biologically Inspired Engineering at Harvard Medical School (HMS), the Professor of Pathology at Brigham and Women’s Hospital, and the Howard Professor of Pathology at Brigham and Women’s Hospital. He is also a professor at the Hughes Medical Institute.
From blood to EV, biomarkers, and diagnosis
Motivated by the diagnostic potential of EVs, especially in the early diagnosis of PD, AD, and other brain disorders, the Walt group has systematically filled in key pieces to this technological jigsaw puzzle.
With philanthropic support from Good Ventures, the Chan Zuckerberg Initiative, and most recently the Michael J. Fox Foundation, they previously developed a technical framework for quantifying EV, and using this quantification We compared EV isolation methods from body fluids.
Their methodology relies on a separation technique known as size exclusion chromatography (SEC), which recovers most EVs from biological fluids, and an ultrahigh-performance technique that allows counting single protein molecules associated with EVs captured and visualized with specific antibodies. It is a combination of the sensitive “Simoa assay”.
To date, the team has designed Simoa assays for various EV-specific biomarkers, importantly excluding the candidate surface protein L1CAM, which is widely used as a target to isolate brain-specific EVs. This resulted in an important course correction for the field. .
“To answer the conceptually simple but technically difficult question of what percentage of certain proteins present in plasma (such as ⍺-synuclein) are inside EVs compared to outside, we We used an SEC isolation method that we previously developed to isolate most EVs from plasma. This was coupled with an optimized “proteinase protection assay” that uses enzymes to isolate EVs surrounded by membranes. Gently and efficiently chews up all proteins from the surface of isolated EVs while leaving the interior intact. ” said co-author Dr. Dima Ter Ovanesyan, a senior scientist at the Wyss Institute and co-first author and co-lead of the EV project with postdoctoral fellow Tal Gilboa, Ph.D.
Also, to measure very low levels of ⍺-synuclein, Dr. Gilboa collaborated with postdoctoral fellow Dr. Gina Wang and Sarah Whiteman, a Wyss research assistant at the Walt Institute, used previously reported assays. We have developed a simour assay for ⍺-synuclein that is much more sensitive than the ⍺-synuclein assay.
Using this assay in their protocol, the research team demonstrated that the majority of ⍺-synuclein in EVs isolated using the SEC protocol was protected, and that this amount was less than 5% of total plasma ⍺-synuclein. I was able to confirm that there is.
Understanding this amount is particularly important for the ultimate goal of measuring ⍺-synuclein in neuron-derived EVs. This is because EVs originating from specific tissues such as the brain are expected to be rare compared to EVs originating from blood cells, which also express ⍺-synuclein. .
Importantly, in addition to the ultrasensitive Simoa assay, which enabled the detection of normal, unmodified ⍺-synuclein protein, we also developed an assay that can detect ⍺-synuclein, which is phosphorylated at a specific site (pSer129) during the process. That’s it. PD progress.
“When we applied our advanced methodology to a cohort of blood samples from patients with PD and Lewy body dementia and healthy control donors, we found that the ratio of phosphorylated ⍺-synuclein to total ⍺-synuclein was 2 to 2. 3, twice as high inside the EV compared to outside the EV,” Gilboa said.
“This was very interesting because it suggests that EVs may be protecting the phosphorylation status of proteins from circulating phosphatases, which would otherwise be very beneficial.” The mark will be erased.”
The research team is currently investigating further whether these assays can be used to differentiate PD patients from PD patients.
“The research by David Walt’s team represents a technological marvel that brings us closer to the next generation of diagnostic platforms with extraordinary potential. “It’s not too long before we can use information-rich cell-derived vesicles as a window into a patient’s brain without the need for surgery,” said Wyss Founding Director Donald Ingber, MD. He also holds 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 University’s John A. Paulson School of Engineering and Applied Sciences.
Additional authors on 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., Ph.D., Parker Family Professor of Neurology at the Perelman School of Medicine. . The University of Pennsylvania, Philadelphia, has worked with Walt’s group since the beginning of the EV project, as has George Kannercutt.
Funding: This research was supported by grants from the Michael J. Fox Foundation (Grant #2021A017224), the Chan Zuckerberg Initiative NeuroDegeneration Challenge Network, and Good Ventures. Gilboa is a recipient of the Weizmann Institute of Science Women Postdoctoral Career Development Award.
About this Parkinson’s disease research news
author: benjamin bettner
sauce: Wyss Institute
contact: Benjamin Bottner – Wyss Institute
image: Image credited to Neuroscience News
Original research: Closed access.
“Measurement of α-synuclein as a protein cargo in plasma extracellular vesicles” by David Walt et al. PNAS
abstract
Measurement of α-synuclein as a protein cargo in plasma extracellular vesicles
Extracellular vesicles (EVs) are released from all cells and have attracted great promise as a type of biomarker. This promise has led to increased interest in measuring EV proteins from both total EVs and brain-derived EVs in plasma.
However, the measurement of EV cargo proteins has been difficult due to the low levels of EVs present and EV isolation methods that are incomplete in separating EVs from free proteins. Therefore, it is difficult to know whether the proteins measured after EV isolation are really present within EVs.
In this study, we developed a method to measure whether proteins are present within EVs and to quantify the proportion of proteins within EVs relative to total plasma.
To achieve this, we combine a high-yield size exclusion chromatography protocol with an optimized protease protection assay and single molecule array (Simoa) digital enzyme-linked immunosorbent assay (ELISA) to detect EVs. We measured the proteins within with ultra-high sensitivity.
We applied these methods to the analysis of α-synuclein and confirmed that a small portion of total plasma α-synuclein is present within EVs. Furthermore, we developed a highly sensitive Simoa assay for phosphorylated α-synuclein (phosphorylated on the Ser129 residue).
We found that the ratio of phosphorylated α-synuclein to total α-synuclein was enriched inside EVs compared to outside EVs.
Finally, we applied the method we developed to measure total and phosphorylated α-synuclein in EVs from patient samples with Parkinson’s disease and Lewy body dementia.
This study provides a framework for determining protein levels of EVs and is an important step in the development of EV diagnostics for diseases of the brain and other organs.
