Researchers achieve accurate EV analysis to detect brain disease

Brain disorders such as Parkinson’s disease (PD) or Alzheimer’s disease (AD) begin to develop in patients much earlier than when their first clinical symptoms appear. Treating patients in these early stages could slow or even stop their disease, but there is currently no way to diagnose brain disorders in these pre-symptomatic stages. Until now, the specific brain lesions caused by, for example, Parkinson’s disease can only be detected by analyzing brain biopsies, which can only be obtained posthumously.

To overcome this critical bottleneck, researchers have pursued the new concept of “liquid biopsies,” in which blood or other body fluids can be easily extracted using non-invasive procedures and analyzed for molecules derived from the brain and other solid substances. tissues. A particularly promising target in body fluids are “extracellular vesicles” (EVs), small membrane-bound sacs released by brain and other cells into the surrounding fluids. These sacs contain a variety of molecules that may be unique to the cell types they produce, such as the brain, and thus may also contain protected biomarkers for the early onset of Parkinson’s disease and other brain diseases.

However, despite recent progress, EV experts have not been able to address the issue of whether certain biomarker molecules they measured in isolated EVs are strictly contained within EVs or non-specifically bound to their surfaces. This challenge has actually prevented them from drawing unequivocal conclusions about cargo molecules in EVs from all types of tissues.

Now a collaborative team led by David Walt, Ph.D. at the Wyss Institute of Harvard University and Brigham and Women’s Hospital (BWH) in Boston solved this problem by adding a crucial step to an already validated ultrasensitive protocol. Enzymatically digesting all surface-bound proteins from a purified EV population allowed them to specifically enter the cargo protected in EVs while eliminating non-specific “contaminants”. Using their improved protocol to measure the PD biomarker ⍺-synuclein in blood, they were able for the first time to accurately determine the small fraction of each protein in EVs versus how much of it is freely present in total blood plasma.

Importantly, they integrated this advance with a newly developed ultrasensitive detection assay for a form of ⍺-synuclein that becomes increasingly phosphorylated during the progression of Parkinson’s disease and the related condition Lewy Body Dementia. By analyzing a cohort of patient samples, they were able to detect an enrichment of the pathological ⍺-synuclein protein in EVs relative to total plasma. The findings were published in PNAS.

“Research on EVs in our and other groups over the past decades has steadily increased our understanding of their complex biology and molecular composition. Yet, isolating pure tissue-specific EVs from body fluids such as blood or the cerebrospinal fluid surrounding the central nervous system, including the brain, and validating and quantifying their actual content with precise measurements still pose formidable technical challenges,” said Walt, Member from the Wyss Core Faculty.

Our recent work provides a solution to help fill this technological gap, and brings us closer to obtaining EVs that are free of contamination so that we can use them as rich sources for clinical biomarkers, as we show with the example of phosphorylated ⍺-synuclein. .”

David Walt, Wyss Institute at Harvard University

Walt is the faculty leader 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 professor at the Howard Hughes Medical Institute.

From blood to EVs to biomarkers to diagnosis

Primarily motivated by the diagnostic promise of EVs for the early diagnosis of Parkinson’s, AD and other brain disorders, the Walt group has systematically filled in vital pieces in this technical puzzle. 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 methodology combines a separation technique known as size exclusion chromatography (SEC) to recover the most EVs from biofluids with ultrasensitive “Simoa assays” that allowed them to count individual protein molecules associated with EVs they captured and visualized with specific antibodies . Meanwhile, the team has developed Simoa assays for a variety of EV-specific biomarkers and, importantly, ruled out a commonly used candidate surface protein, L1CAM, as a target to isolate brain-specific EVs, providing the field with an important course correction. .

“To answer the conceptually simple but technically challenging question of what percentage of a particular protein (such as ⍺-synuclein) present in plasma is contained in EVs relative to the outside, we used SEC isolation methods that we have previously developed to isolate the most EVs from plasma along with an optimized ‘proteinase protection assay’ where we use an enzyme to gently but efficiently chew all proteins from the surface of isolated EVs, while leaving the membrane-enclosed EV interior intact.” said co-first author Dima Ter-Ovanesyan, Ph.D., who is a senior scientist at the Wyss Institute and along with co-first author and postdoctoral fellow Tal Gilboa, Ph.D. leads the EV project.

To measure ⍺-synuclein at very low levels, Gilboa, along with postdoctoral fellow Gina Wang, Ph.D. and Wyss research assistant Sara Whiteman in the Walt lab developed a Simoa test for ⍺-synuclein that is much more sensitive than any previously reported test. 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 represented less than 5% of the total ⍺-synuclein in blood plasma . Understanding this quantity is especially important for the ultimate goal of measuring ⍺-synuclein in neuron-derived EVs, as EVs originating from a specific tissue such as the brain are expected to be rare compared to EVs from blood cells, where ⍺-synuclein is also expressed. .

Importantly, in addition to their ultrasensitive Simoa assay that allowed them to detect the normal, unmodified ⍺-synuclein protein, they also developed an assay capable of detecting ⍺-synuclein expressed at a specific site (pSer129) in the becomes phosphorylated over time. of PD progression. “When we applied our advanced methodology to a cohort of blood samples obtained from patients with Parkinson’s disease and Lewy Body Dementia, as well as healthy control donors, we found that the ratio of phosphorylated ⍺-synuclein to total ⍺-synuclein was two to three. fold higher inside EVs relative to outside EVs,” says Gilboa. “This was extremely exciting because it suggests that EVs may protect the phosphorylation state of proteins from circulating phosphatases that would otherwise erase this highly informative mark.” tests can be used to distinguish Parkinson’s patients from people without the disease.

“The work of David Walt’s team presents a technological tour de force that brings us ever 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 see into patients’ brains without the need for surgery,” said Wyss Founding Director Donald Ingber, MD , Ph.D., who also holds the Judah Folkman professor of vascular biology at HMS and Boston Children’s Hospital and the Hansjörg Wyss professor of biologically inspired technology at Harvard’s John A. Paulson School of Engineering and Applied Sciences.