Objective:

Optimize exosomal isolation and lysis from plasma to evaluate alpha-synuclein as a biomarker distinguishing Parkinson’s disease (PD) from multiple system atrophy (MSA).

Background:

Exosomes are nanoscale extracellular vesicles (EVs) that mediate intercellular communication and reflect their cells of origin. They can be isolated from accessible biofluids, making them promising biomarker candidates. Alpha-synuclein, a presynaptic neuronal protein contained in exosomes, is normally monomeric but aggregates in neurodegenerative disease. In PD, it accumulates in neuronal Lewy bodies, whereas in MSA it forms glial cytoplasmic inclusions. Current antemortem diagnostic accuracy for PD is around 80%, with higher misdiagnosis rates for MSA. Fluid-based biomarkers could substantially improve diagnostic certainty and patient care.

Design/Methods:

Plasma was collected from PD and non-PD patients under VA IRB approval.  EVs were isolated from plasma using three methods: TEI isolation kit, ultracentrifugation (UC), and polyethylene glycol (PEG) precipitation. EV size and concentration were determined with ZetaView nanoparticle tracking analysis. Lysis conditions tested included RIPA buffer, Tris–Triton buffer, sonication, and combined RIPA + sonication. Protein yield was quantified by BCA assay, and Western blot probed for exosomal markers (Alix, CD63, CD81) and alpha-synuclein.

Results:

PEG yielded the greatest EV recovery, followed by TEI, then UC. ZetaView analysis confirmed recovery of particles within 30-150nm for PEG (3.4 × 10^6 particles/mL), TEI (3.0 × 10^6 particles/mL), and UC (1.74 × 10^6 particles/mL). BCA quantification showed the highest protein concentration with RIPA + sonication (0.55 mg/mL). Western blot demonstrated enrichment of exosomal markers and robust alpha-synuclein detection in PEG-isolated exosomes.

Conclusions:

PEG precipitation with RIPA + sonication lysis provides an effective workflow for isolating and analyzing alpha-synuclein–containing EVs. Extending this approach to saliva and urine and applying RT-QuIC assays to quantify misfolded alpha-synuclein may yield reliable biomarkers for differentiating PD from MSA and improving diagnostic accuracy in synucleinopathies.