Detection of skin α-synuclein using RT-QuIC as a diagnostic biomarker for Parkinson's disease in the Chinese population

Abstract

Background: Several studies have indicated that skin holds promise as a potential sample for detecting pathological α-Syn and serving as a diagnostic biomarker for α-synucleinopathies. Despite reports in Chinese PD patients, comprehensive research on skin α-Syn detection using RT-QuIC is lacking.

Objective: This study aimed to evaluate the diagnostic performance of skin samples using RT-QuIC from PD patients in the Chinese population.

Methods: Patients with sporadic PD and controls were included according to the British PD Association Brain Bank diagnostic criteria. The seeding activity of misfolded α-Syn in these skin samples was detected using the RT-QuIC assay after protein extraction. Biochemical and morphological analyses of RT-QuIC products were conducted by atomic force microscopy, transmission electron microscopy, Congo red staining, and dot blot analysis.

Result: 30 patients clinically diagnosed with PD and 28 controls with non-α-synucleinopathies were included in this study. 28 of 30 PD patients demonstrated positive α-Syn seeding activity by RT-QuIC assay. In contrast, no α-Syn seeding activity was detected in the 28 control samples, with an overall sensitivity and specificity of 93.3% and 100%, respectively (P < 0.001). Biochemical characterization of the RT-QuIC product indicated fibrillary α-Syn species in PD-seeded reactions, while control samples failed in the conversion of recombinant α-Syn substrate.

Conclusion: This study applied RT-QuIC technology to identify misfolded α-Syn seeding activity in skin samples from Chinese PD patients, demonstrating high specificity and sensitivity. Skin α-Syn RT-QuIC is expected to be a reliable approach for the diagnosis of PD.

Keywords: Parkinson’s disease; RT-QuIC; Skin; α-synuclein.

Fig. 1

Detection of α-Syn seeding activity by skin RT-QuIC assay in patients with PD and controls. A RT-QuIC detection of α-Syn seeding activity in the cervical skin homogenates from 4 patients with PD and 4 controls. B RT-QuIC detection of α-Syn seeding activity in the skin homogenates of 30 patients and 28 controls. C Scatter graph of maximal thioflavin T (ThT) fluorescence at 70 h of the α-Syn seeding activity by RT-QuIC assay. D ROC curve and AUC calculation for comparison of α-Syn seeding activity between patients with PD and controls (P < 0.001)

Fig. 2

Detection of fibrillary α-Syn in the RT-QuIC product of PD patients. A, B The structural features of α-Syn aggregates extracted from PD-seeded RT-QuIC reactions were analyzed by employing transmission electron microscopy (TEM) and atomic force microscopy (AFM), which could not be observed in the control samples. C Congo red staining, observed under polarized light, exhibited an apple-green color in PD patients’ samples obtained from the RT-QuIC assay, whereas the control samples without PD showed a negative result. The scale bar represents 200 μm. D Analysis of Dot Blot both T0 and RT-QuIC Reaction in PD and Control Groups: Dot blot analysis was conducted on skin samples from three PD and three controls at both T0 and after RT-QuIC reactions using α-Syn filament conformation-specific antibodies (top panel) and total α-Syn antibodies (bottom panel). Densitometric quantification of α-Syn filament conformation-specific and total α-Syn levels revealed significantly increased levels of α-Syn filaments for the PD group compared to the control group, while total αSyn levels did not differ between groups. Results were analyzed using Student’s two-sample t-test. **p < 0.01 and not significant (ns) when P > 0.05

Fig. 3

Skin RT-QuIC results exhibit no correlation with disease progression. Raw RT-QuIC assay parameters reveal no association with PD duration: longer disease duration (A, E), higher Hoehn and Yahr (H&Y) stage (B, F), higher Unified Parkinson’s Disease Rating Scale (UPDRS) score (C, G), and lower Mini-Mental State Examination (MMSE) score (D, H) do not exhibit a higher final ThT fluorescence or longer lag phase