Seeding activities of disease-associated α-synuclein aggregates (αSynD), a hallmark of Parkinson’s disease (PD), are detectable by seed amplification assay (αSyn-SAA) and being developed as a diagnostic biomarker for PD. Sensitive and accurate αSyn-SAA for blood or saliva would greatly facilitate PD diagnosis. This prospective diagnostic study conducted αSyn-SAA analyses on serum and saliva samples collected from patients clinically diagnosed with PD or healthy controls (HC). 124 subjects (82 PD, 42 HC) donated blood and had extensive clinical assessments, of whom 74 subjects (48 PD, 26 HC) also donated saliva at the same visits. An additional 57 subjects (35 PD, 22 HC) donated saliva and had more limited clinical assessments. The mean ages were 69.21, 66.55, 69.58, and 64.71 years for PD serum donors, HC serum donors, PD saliva donors, and HC saliva donors, respectively. αSynD seeding activities in either sample type alone or both sample types together were evaluated for PD diagnosis. Serum αSyn-SAA data from 124 subjects showed 80.49% sensitivity, 90.48% specificity, and 0.9006 accuracy (AUC of ROC); saliva αSyn-SAA data from 131 subjects attained 74.70% sensitivity, 97.92% specificity, and 0.8966 accuracy. Remarkably, the combined serum and saliva αSyn-SAA from 74 subjects with both sample types achieved better diagnostic performance: 95.83% sensitivity, 96.15% specificity, and 0.98 accuracy. In addition, serum αSynD seeding activities correlated inversely with Montreal Cognitive Assessment in males and positively with Hamilton Depression Rating Scale in females and in the < 70 age group, whereas saliva αSynD seeding activities correlated inversely with age at diagnosis in males and in the < 70 age group. Our data indicate that serum and saliva αSyn-SAA together can achieve high diagnostic accuracy for PD comparable to that of CSF αSyn-SAA, suggesting their potential utility for highly sensitive, accurate, and minimally invasive diagnosis of PD in routine clinical practice and clinical studies.

Background: Tauopathies are a group of age-related neurodegenerative diseases characterized by the accumulation of pathologically hyperphosphorylated tau protein in the brain, leading to prion-like aggregation and propagation. They include Alzheimer’s disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick’s disease (PiD). Currently, reliable diagnostic biomarkers that directly reflect the capability of propagation and spreading of misfolded tau aggregates in peripheral tissues and body fluids are lacking. Methods: We utilized the seed-amplification assay (SAA) employing ultrasensitive real-time quaking-induced conversion (RT-QuIC) to assess the prion-like seeding activity of pathological tau in the skin of cadavers with neuropathologically confirmed tauopathies, including AD, PSP, CBD, and PiD, compared to normal controls. Results: We found that the skin tau-SAA demonstrated a significantly higher sensitivity (75–80%) and specificity (95–100%) for detecting tauopathy, depending on the tau substrates used. Moreover, the increased tau-seeding activity was also observed in biopsy skin samples from living AD and PSP patients examined. Analysis of the end products of skin-tau SAA confirmed that the increased seeding activity was accompanied by the formation of tau aggregates with different physicochemical properties related to two different tau substrates used. Conclusions: Overall, our study provides proof-of-concept that the skin tau-SAA can differentiate tauopathies from normal controls, suggesting that the seeding activity of misfolded tau in the skin could serve as a diagnostic biomarker for tauopathies.

Alpha-synuclein seed amplification assays (αSyn-SAAs) have emerged as promising diagnostic tools for Parkinson’s disease (PD) by detecting misfolded αSyn and amplifying the signal through cyclic shaking and resting in vitro. Recently, our group and others have shown that multiple biospecimens, including CSF, skin, and submandibular glands (SMGs), can be used to seed the aggregation reaction and robustly distinguish between patients with PD and non-disease controls. The ultrasensitivity of the assay affords the ability to detect minute quantities of αSyn in peripheral tissues, but it also produces various technical challenges of variability. To address the problem of variability, we present a high-yield αSyn protein purification protocol for the efficient production of monomers with a low propensity for self-aggregation. We expressed wild-type αSyn in BL21 Escherichia coli, lysed the cells using osmotic shock, and isolated αSyn using acid precipitation and fast protein liquid chromatography (FPLC). Following purification, we optimized the ionic strength of the reaction buffer to distinguish the fluorescence maximum (Fmax) separation between disease and healthy control tissues for enhanced assay performance. Our protein purification protocol yielded high quantities of αSyn (average: 68.7 mg/mL per 1 L of culture) and showed highly precise and robust αSyn-SAA results using brain, skin, and SMGs with inter-lab validation.

Synucleinopathies, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), share pathological accumulation of misfolded α-synuclein (αSyn) in the brain and overlapping clinical features, complicating accurate diagnosis with current methods. In this study, we utilized a real-time quaking-induced conversion (RT-QuIC) assay to demonstrate that autopsied skin samples from PD, DLB, and MSA patients (but not non-synucleinopathy controls) seed aggregation of recombinant αSyn. While RT-QuIC generated similarly positive fluorescence kinetic curves across synucleinopathies, biochemical and morphological analyses of RT-QuIC end products revealed distinct properties in the resulting αSyn aggregates. Notably, αSyn aggregates from DLB samples exhibited the highest resistance to proteinase K digestion, whereas MSA-derived aggregates showed the least aggregated bands on Western blots. Transmission electron microscopy revealed significant differences in length, width, and volume of skin αSyn fibrils of RT-QuIC end products from different synucleinopathies. These findings provide critical insights into disease-specific αSyn structural characteristics and suggest new strategies to improve diagnostic discrimination.

Background Tauopathies are a group of age-related neurodegenerative diseases characterized by the accumulation of pathologically phosphorylated tau protein in the brain, leading to prion-like propagation and aggregation. They include Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick's disease (PiD). Currently, reliable diagnostic biomarkers that directly reflect the capability of propagation and spreading of misfolded tau aggregates in peripheral tissues and body fluids are lacking. Methods We utilized the seed-amplification assay (SAA) employing ultrasensitive real-time quaking-induced conversion (RT-QuIC) to assess the prion-like seeding activity of pathological tau in the skin of cadavers with neuropathologically confirmed tauopathies, including AD, PSP, CBD, and PiD, compared to normal controls. Results We found that the skin prion-SAA demonstrated a significantly higher sensitivity (75–80%) and specificity (95–100%) for detecting tauopathy, depending on the tau substrates used. Moreover, increased tau-seeding activity was also observed in biopsy skin samples from living AD and PSP patients examined. Analysis of the end products of skin-tau SAA confirmed that the increased seeding activity was accompanied by the formation of tau aggregates with different physicochemical properties related to two different tau substrates used. Conclusions Overall, our study provides proof-of-concept that the skin tau-SAA can differentiate tauopathies from normal controls, suggesting that the seeding activity of misfolded tau in the skin could serve as a diagnostic biomarker for tauopathies.

Importance: Parkinson’s disease (PD), the second most common neurodegenerative disease, is pathologically characterized by intraneuronal deposition of misfolded alpha-synuclein aggregates (αSyn ^D ). αSyn ^D seeding activities in CSF and skin samples have shown great promise in PD diagnosis, but they require invasive procedures. Sensitive and accurate αSyn ^D seed amplification assay (αSyn-SAA) for more accessible and minimally invasive samples (such as blood and saliva) are urgently needed for PD pathological diagnosis in routine clinical practice. Objective: To develop a sensitive and accurate αSyn-SAA biomarker using blood and saliva samples for sensitive, accurate and minimally invasive PD diagnosis. Design, Setting, and Participants: This prospective diagnostic study evaluates serum and saliva samples collected from patients clinically diagnosed with PD or healthy controls (HC) without PD at an academic Parkinson’s and Movement Disorders Center from February 2020 to March 2024. Patients diagnosed with non-PD parkinsonism were excluded from this analysis. A total of 124 serum samples (82 PD and 42 HC) and 131 saliva samples (83 PD and 48 HC) were collected and examined by αSyn-SAA. Out of the 124 serum donors, a subset of 74 subjects (48 PD and 26 HC) also donated saliva samples during the same visits. PD patients with serum samples had a mean age of 69.21 years (range 44-88); HC subjects with serum samples had a mean age of 66.55 years (range 44-81); PD patients with saliva samples had a mean age of 69.58 years (range 49-87); HC subjects with saliva samples had a mean age of 64.71 years (range 30-81). Main Outcomes and Measures: Serum and/or saliva αSyn ^D seeding activities from PD and HC subjects were measured by αSyn-SAA using the Real-Time Quaking-Induced Conversion (RT-QuIC) platform. These PD patients had extensive clinical assessments including MDS-UPDRS. For a subset of PD and HC subjects whose serum and saliva samples were both collected during the same visits, the αSyn ^D seeding activities in both samples from the same subjects were examined, and the diagnostic accuracies for PD based on the seeding activities in either sample alone or both samples together were compared. Results: RT-QuIC analysis of αSyn ^D seeding activities in the 124 serum samples revealed a sensitivity of 80.49%, a specificity of 90.48%, and an accuracy of 0.9006 (AUC of ROC, 95% CI, 0.8472-0.9539, p <0.0001) for PD diagnosis. RT-QuIC analysis of αSyn ^D seeding activity in 131 saliva samples revealed a sensitivity of 74.70%, a specificity of 97.92%, and an accuracy of 0.8966 (AUC of ROC, 95% CI, 0.8454-0.9478, p <0.0001). When aSyn ^D seeding activities in the paired serum-saliva samples from the subset of 48 PD and 26 HC subjects were considered together, sensitivity was 95.83%, specificity was 96.15%, and the accuracy was 0.98 (AUC of ROC, 95% CI, 0.96-1.00, p <0.001), which are significantly better than when αSyn ^D seeding activities in either serum or saliva were used alone. For the paired serum-saliva samples, when specificity was set at 100% by elevating the αSyn-SAA cutoff values, a sensitivity of 91.7% and an accuracy of 0.9457 were still attained. Detailed correlation analysis revealed that αSyn ^D seeding activities in the serum of PD patients were correlated inversely with Montreal Cognitive Assessment (MoCA) score ( p =0.04), positively with Hamilton Depression Rating Scale (HAM-D) ( p =0.03), and weakly positively with PDQ-39 cognitive impairment score ( p =0.07). Subgroup analysis revealed that the inverse correlation with MoCA was only seen in males ( p =0.013) and weakly in the ≥70 age group ( p =0.07), and that the positive correlation with HAM-D was only seen in females ( p =0.04) and in the <70 age group ( p =0.01). In contrast, αSyn ^D seeding activities in the saliva of PD patients were inversely correlated with age at diagnosis ( p =0.02) and the REM sleep behavior disorder (RBD) status ( p =0.04), but subgroup analysis showed that the inverse correlation with age at diagnosis was only seen in males ( p =0.04) and in the <70 age group ( p =0.01). Conclusion and Relevance: Our data show that concurrent RT-QuIC assay of αSyn ^D seeding activities in both serum and saliva can achieve high diagnostic accuracies comparable to that of CSF αSyn-SAA, suggesting that αSyn ^D seeding activities in serum and saliva together can potentially be used as a valuable biomarker for highly sensitive, accurate, and minimally invasive diagnosis of PD in routine clinical practice. αSyn ^D seeding activities in serum and saliva of PD patients correlate differentially with some clinical characteristics and in an age and sex-dependent manner. KEY POINTS: Question: Are αSyn ^D seeding activities in serum and saliva together a more sensitive and accurate diagnostic PD biomarker than αSyn ^D seeding activities in either sample type alone? Are αSyn ^D seeding activities in either serum or saliva correlated with any clinical characteristics? Findings: Examinations of αSyn ^D seeding activities in 124 serum samples and 131 saliva samples from PD and heathy control subjects show that αSyn ^D seeding activities in both serum and saliva samples together can provide significantly more sensitive and accurate diagnosis of PD than either sample type alone. αSyn ^D seeding activities in serum or saliva exhibit varied inverse or positive correlations with some clinical features in an age and sex-dependent manner. Meaning: αSyn ^D seeding activities in serum and saliva together can potentially be used as a valuable pathological biomarker for highly sensitive, accurate, and minimally invasive PD diagnosis in routine clinical practice and clinical studies, and αSyn ^D seeding activities in serum or saliva correlate with some clinical characteristics in an age and sex-dependent manner, suggesting some possible clinical utility of quantitative serum/saliva αSyn-SAA data.