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Serum Neuronal Extracellular Vesicles for RT-QuIC Assays to Detect Pathological α-Synuclein in Synucleinopathies
Hye Joung Choi1,2,3, Dong Gyun Ko1,2,3,4, JeKuk Yu1,2,3,4
1Department of Neurology, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Korea.
Objective:
Pathological α-synuclein aggregation is a key finding in synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. The real-time quaking-induced conversion (RT-QuIC) assay using cerebrospinal fluid (CSF) can sensitively detect pathological α-synuclein aggregates and is supported by a strong biological rationale. However, the invasive nature of CSF collection limits its clinical utility. A blood-based RT-QuIC assay is therefore of growing interest; although evidence remains limited, it has shown good performance in distinguishing patients with PD from healthy controls (HCs). In this study we investigated pathological α-synuclein aggregates in the neuron-derived extracellular vesicles (nEVs) isolated from serum samples of patients with synucleinopathies and HCs.
Methods:
Serum samples were collected from patients diagnosed with synucleinopathies and HCs without neurological disorders. Total extracellular vesicles (EVs) were isolated from serum using an ExoQuick kit, after which nEVs were isolated via L1-cell adhesion molecule immunocapture. The identity of the nEVs was confirmed by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. Pathological α-synuclein aggregates in EVs were assessed by western blotting, dot blotting, and RT-QuIC assays.
Results:
The concentration and abundance of EVs were comparable between the PD and HC groups. TEM and NTA confirmed EV morphology and size distribution, and western blotting validated the neuronal quality of the nEVs. Compared with those from HCs, nEVs from patients with synucleinopathies showed higher levels of phosphorylated or aggregated α-synuclein. Optimized RT-QuIC conditions using nEV enabled preliminary discrimination of synucleinopathy from HC with nEVs, with distinct kinetic profiles observed between groups.
Conclusion:
Serum-derived nEVs represent a promising, minimally invasive seed source for RT-QuIC assays, offering robust diagnostic performance for the detection of synucleinopathies and potential applicability in broader clinical settings.
