NEW YORK – A team led by scientists at Purdue University and Tymora Analytical Operations has identified a set of protein biomarkers that could prove useful for diagnosing Parkinson's disease.
The researchers identified the markers using mass spec analysis of proteins and phosphoproteins contained in extracellular vesicles (EV) collected from urine, detailing their work in a study published this month in Communications Medicine.
The study was partially funded by the Michael J. Fox Foundation for Parkinson's Research as part of an effort by the organization to explore if analysis of EVs could identify novel biomarkers for the disease. Shed by cells into various bodily fluids, EVs are thought to reflect the molecular make-up of their cells of origin. This has made them an area of interest in liquid biopsy research, the idea being that it might be easier to measure proteins or nucleic acids in exosomes derived from, for instance, cancer cells, than to detect cancer-linked nucleic acids or proteins circulating freely in patient blood or urine.
The same thinking applies to neurodegenerative conditions, and particularly to efforts to identify markers for these conditions in sample sources like blood or urine, which are more easily accessible than cerebrospinal fluid but typically contain much lower concentrations of the relevant markers.
West Lafayette, Indiana-based Tymora is a spinout from the lab of Purdue University researcher Andy Tao, a professor of chemical biology and analytical chemistry and, along with Tymora CEO Anton Iliuk, an author on the Communications Medicine paper. Tymora specializes in proteomic and phosphoproteomic analysis of EVs, which it sells as a research service to outside partners and uses for its own internal biomarker and diagnostics development work.
In 2018, the company and collaborators published a study in the Journal of Proteome Research in which they identified roughly 860 phosphoproteins and more than 2,000 unmodified proteins in EVs collected from urine. Researchers at the Michael J. Fox Foundation came across the study and were "intrigued by the results," which included the detection of several Parkinson's-linked proteins, said Shalini Padmanabhan, VP of research programs at the foundation and an author on the Communications Medicine paper.
The foundation, Padmanabhan noted, had at that time assembled a large set of urine samples from Parkinson's patients and saw in Tymora's technology "an opportunity to test a novel approach to identify[ing] changes in protein signatures in EVs from [Parkinson's] patients compared to healthy controls."
The researchers used Tymora's EVtrap technology to isolate EVs from 82 urine samples (21 healthy controls, 13 healthy carriers of the Parkinson's-linked mutation LRRK2, 28 Parkinson's patients without the LRRK2 mutation, and 20 Parkinson's patients with the LRRK2 mutation) collected at Columbia University Irving Medical Center. The EVtrap approach uses magnetic beads functionalized with lipophilic and hydrophilic groups that bind to the lipid bilayer membranes that encapsulate EVs. The approach enables sensitive and reproducible capture of EVs while limiting capture of high-abundance circulating proteins, an issue that has confounded some other approaches to EV enrichment.
Following isolation of the EVs, the researchers analyzed their protein contents via LC-MS on a Thermo Fisher Scientific Q-Exactive HF-X instrument. They identified 4,476 unique proteins and 2,680 unique phosphoproteins from which they identified a list of 48 potential markers that they then winnowed down to a list of six top performers. They found that the six-marker panel was able to distinguish between healthy individuals and those with Parkinson's disease with an area under the curve of .94.
The researchers then validated these top-performing markers along with other proteins known to be involved in Parkinson's disease in two experiments, one using targeted mass spec to measure them in a set of 13 healthy controls and 23 individuals with Parkinson's disease, and another using immunoassays to measure them in a set of 10 healthy controls and 10 individuals with Parkinson's.
Tao said his lab continues to collaborate with the Columbia University group to access additional samples. He is also working with his Purdue colleague Jean-Christophe Rochet whose research is focused on the role of protein aggregation in neurodegenerative diseases including Parkinson's, Alzheimer's, and dementia with Lewy bodies.
One of the questions Tao and Rochet are exploring in that work is whether EVs may be a useful source of alpha-synuclein (αSyn), a neuronal protein linked to Parkinson's and other neurological conditions. In Parkinson's patients, misfolded versions of aggregated αSyn accumulate throughout the brain in clumps called Lewy bodies that are believed to cause neuronal damage. The protein is considered both a potential drug target and a biomarker for the disease.
Alpha-synuclein seed amplification assays, in which αSyn from a patient is incubated with normal αSyn and observed to see if it produces the aggregates characteristic of Parkinson's disease, is one of the more promising approaches to diagnosing the condition. Typically, αSyn samples are collected in patient cerebrospinal fluid, which requires a spinal tap. This has led researchers to explore if the protein might be collected less invasively, via blood or urine samples, for instance, with EVs one potential sampling route.
Padmanabhan noted that "while the full extent of synuclein distribution and its relevance to [Parkinson's] biology remains incompletely understood, it has been proposed that EVs may be enriched for synuclein, including pathological forms."
She added that, to date, the Fox Foundation's main efforts on using EVs as a sample source for αSyn have focused on EVs in blood, "where the presence of synuclein species is better established." She said, though, that the organization "continues to explore all potential alternatives to CSF, towards an improved assay for clinical use as part of our ongoing programs to better understand synuclein biology."
Tymora does not itself plan to pursue further development of the markers identified in the Communications Medicine paper, said CEO Iliuk, but he said that neurodegenerative diseases, and Alzheimer's disease, specifically, have become a focus of both Tymora's internal biomarker development work and its work for outside clients.
Iliuk noted that while plasma is widely seen as the most viable alternative to CSF for Alzheimer's biomarker testing, the Parkinson's research demonstrates the potential of urine EVs as a source of biomarkers for neurodegenerative disease.
"We have done quite a bit of work in plasma, and that is I think where the primary focus has been," he said. "But urine is something we have recently been looking at. It is very early stages, and there is a lot of hesitation to see it as a viable biofluid because it is so far away from the brain and so it isn't an obvious choice. But I think the Parkinson's study shows that [signs of] neurodegenerative conditions can travel as far as the urine and be detected."
The Fox Foundation has supported a number of other efforts looking for protein markers for Parkinson's disease in urine, including proteomics work published in 2021 by the lab of Matthias Mann, head of the department of proteomics and signal transduction at the Max Planck Institute of Biochemistry, that identified several potential protein markers for the disease.