Abnormal α-synuclein aggregation is a key pathological feature of synucleinopathies, a group of neurodegenerative diseases including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Recent studies have demonstrated that these diseases involve not only the brain but also peripheral nerves, leading to the hypothesis that a blood-mediated pathway might be involved in systemic α-synuclein spread. To address this question, we developed a method to detect trace amounts of aggregated α-synuclein in the serum by modifying the Real-Time Quaking-induced Conversion (RT-QuIC) method, used in prion disease diagnosis. Although cerebrospinal fluid (CSF)-based RT-QuIC methods reportedly exist, lumbar puncture is invasive, technically challenging, and time-consuming, often causing post-procedure headaches. Applying this approach to all cases is thus difficult.
In synucleinopathies, pathological α-synuclein aggregates could act as templates to convert endogenous normal α-synuclein into pathological forms. However, α-synuclein levels in the blood are extremely low, making detection challenging. We attempted to enhance RT-QuIC sensitivity for detecting trace amounts of abnormal α-synuclein aggregates circulating in the serum of patients with synucleinopathy. We performed immunoprecipitation (IP) followed by RT-QuIC, thereby amplifying abnormal protein structures in vitro, using them as seeds for aggregation reactions. We monitored the amplification real-time by measuring Thioflavin T (ThT) fluorescence intensity, labeling β-sheet structures through specific binding.
In prion disease diagnosis, CSF is directly used as a seed. However, using blood samples without any treatment failed to detect misfolded protein detection via RT-QuIC. Therefore, we performed IP on patient blood samples using magnetic beads. We used an anti-α-synuclein antibody (MJFR1) to concentrate α-synuclein. The amplification reaction involved two stages: 1) reaction mixture incubation with IP-serum to enable healthy and misfolded α-synuclein co-aggregation and 2) promoting healthy-to-misfolded conversion, detected by ThT. We optimized the reaction conditions to achieve a maximum fluorescence intensity of 260,000 RFU, allowing for the distinction between healthy control participants and patients.
In this study, we investigated 270 patients with α-synucleinopathy, 55 non-α-synucleinopathy patients, 128 healthy control participants, 17 patients with Parkin-linked PD (PRKN), and nine patients with REM sleep behavior disorder (RBD). Using IP /RT-QuIC, we detected significantly more prevalent α-synuclein seed presence in the α-synucleinopathy groups. Our Receiver Operating Characteristic analysis confirmed the high diagnostic capacity of this method. In those cases when the diagnosis could be confirmed pathologically, the Lewy body disease, MSA, and control detection rates yielded 100%, 33%, and 0%, respectively. In a collaborative study with the University of Luxembourg involving serum samples from 20 and 15 patients with PD and MSA, respectively, as well as from 20 control participants, the misfolded α-synuclein seed detection rates yielded 75%, 53%, and 5.0%, respectively.
Furthermore, our electron microscopic imaging revealed distinct structures of amplified α-synuclein aggregates in PD/DLB and MSA. We also examined the properties of these aggregates as templates in cultured cells. We could observe different aggregate types, suggesting the potential for disease-specific diagnosis. Finally, we injected amplified patient serum-derived α-synuclein seeds into the striatum of wild-type mice. The results suggested that these seeds retained the ability to induce disease-specific pathologies.
However, the IP/RT-QuIC method has limitations, including variability in seeding ability between the substrate batches and quantitative assessment difficulties due to varying parameters (T1/2, T max, etc.) even when using the same sample. Therefore, future research efforts should focus on improving the stability and quantifiability of the approach. In addition, enhancing detection sensitivity to identify pre-symptomatic conditions like RBD would be necessary. We also aim at elucidating the underlying pathogenesis by focusing on differences in aggregate structures between diseases.