Detection of α‐Synuclein in Oral Mucosa by Seed Amplification Assay in Synucleinopathies and Isolated REM Sleep Behavior Disorder

Objective Evidence of abnormal α‐synuclein (α‐Syn) deposition in the brain is required for definitive diagnosis of synucleinopathies, which remains challenging. The seed amplification assay (SAA) is an innovative technique that can detect the seeding activity of misfolded α‐Syn, enabling the amplification and detection of minute quantities of pathogenic α‐Syn aggregates. This study aimed to evaluate oral mucosa α‐Syn SAA as possible diagnostic and prodromal biomarkers for synucleinopathies. Methods A total of 107 Parkinson's disease (PD) patients, 99 multiple system atrophy (MSA) patients, 33 patients with isolated rapid eye movement sleep behavior disorder (iRBD) and 103 healthy controls (HC) were included. The SAA was applied to detect the seeding activity of α‐Syn from oral mucosa. A combination of morphological, biochemical, and biophysical methods was also used to analyze the fibrils generated from the oral mucosa α‐Syn SAA. Results Structured illumination microscopy images revealed the increased α‐Syn species in oral mucosa of PD, MSA, and iRBD patients than in HCs. Oral mucosa α‐Syn SAA distinguished patients with PD from HC with 67.3% sensitivity and 90.3% specificity. Oral mucosa was α‐Syn SAA positive in 53.5% MSA patients and 63.6% iRBD patients. Furthermore, the α‐Syn fibrils generated from MSA demonstrated greater resistance to proteinase K digestion and exhibited stronger cytotoxicity compared to those from PD patients. Conclusion Oral mucosa α‐Syn seeding activity may serve as novel non‐invasive diagnostic and prodromal biomarkers for synucleinopathies. The α‐Syn aggregates amplified from the oral mucosa of PD and MSA exhibited distinct biochemical and biophysical properties.