Salivary α-Synuclein RT-QuIC Correlates with Disease Severity in de novo Parkinson's Disease.

Abstract

The molecular pathology underlying Parkinson's disease (PD) is characterized by α-synuclein (a-syn) misfolding and aggregation,1 beginning with the formation of a-syn oligomers. A-syn oligomers are responsible for exerting neurotoxicity2 and for driving the propagation of pathology through intercellular transmission and seeded aggregation.3 In recent years, the mechanism of a-syn aggregation has been compared with that of prion proteins and recent studies have used real-time quaking induced conversion (RT-QuIC) or protein misfolding cyclic amplification (PMCA) to detect a-syn oligomers in cerebrospinal fluid,4 submandibular glands,5 skin biopsies,6 or nasal mucosa.7 Here, we exploited the prion-like properties of a-syn aggregates to detect the presence of seeding-competent a-syn oligomers in saliva, an easily accessible biofluid in which increased concentration of a-syn aggregates have been detected in PD patients.8, 9 We have applied RT-QuIC to a cohort of 37 de novo PD patients and 23 sex- and age-matched healthy subjects (HS), and we have analyzed and compared different kinetic parameters including: lag-phase; rate of change; area under the curve (AUC); Thioflavin-T maximum value (Vmax); and percent increase of Thioflavin-T fluorescence from baseline. Detailed methods are provided in Supplementary Appendix S1. Salivary samples derived from PD patients exhibit a greater seeding-capacity in the a-syn RT-QuIC assay compared to salivary samples from HS (Fig. 1A,B, and Bi). Of 37 PD salivary samples, 31 (86%) reached the fluorescence threshold and were, therefore, deemed RT-QuIC positive, with the remaining 6 (14%) being RT-QuIC negative. Among the 23 HS samples, 5 (22%) were deemed RT-QuIC positive and the remaining 18 (78%) RT-QuIC negative. The average lag-phase was significantly shorter in PD samples than in HS samples, whereas the other kinetic parameters were all significantly higher in PD samples relative to HS (Supplementary Fig. S1 and Supplementary eTable S1). To evaluate the capability of the RT-QuIC kinetic parameters to distinguish between PD and HS subjects, we used principal components analysis (PCA) and we found a segregation of PD patient and HS across the PC1 axis (Supplementary Fig. S2). Therefore, to evaluate the diagnostic potential of our RT-QuIC assay we extracted the PC1 Eigenvalue—that represents a composite value of all RT-QuIC parameters submitted to PCA—for each subject and performed receiver operating characteristic (ROC) analysis. We found that salivary RT-QuIC possessed good diagnostic accuracy, with sensitivity of 83.78% (95% confidence interval [CI], 68.86–92.35), specificity of 82.61% (95% CI, 62.86–93.02), and a likelihood ratio of 4.818 (Fig. 1C). When we immunodepleted saliva of both PD patients and HS from a-syn, we detected an increased lag-phase and a significantly decreased Thioflavin-T-Vmax in PD patients, whereas we did not detect any difference in HS (Supplementary Fig. S3), therefore, supporting the specificity of the assay for the a-syn aggregates. We finally aimed to correlate RT-QuIC kinetic parameters with the different clinical scores of PD patients. Z-scores for all clinical and RT-QuIC kinetic parameters were calculated and plotted on a heatmap. Performing agglomerative hierarchical clustering we identified that the clusters with the three principal clinical scores: The Movement Disorder Society-Sponsored Revision of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS), The The Non Motor Symptoms Score (NMSS), and the sum MDS-UPDRS/NMSS were more closely associated to the cluster enriched by RT-QuIC kinetic parameters (Fig. 1D). Interestingly, performing least squares regression we detected a significant positive correlation between summed MDS-UPDRS part III and NMSS and some individual RT-QuIC kinetic parameters (Fig. 1E), indicating that increased disease severity is significantly associated with a greater response in the salivary RT-QuIC assay. A detailed discussion of the data is reported in Supplementary Appendix S1. We thank Dr. Aviva Tolkovsky for continuous helpful and constructive comments about the manuscript and the methodological approach. All authors have no financial disclosures to report. All authors have no financial disclosures to report. (1) Research project: A. Conception, B. Organization, C. Execution; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript: A. Writing of the First Draft, B. Review and Critique. 1A-B: G.V., A.S., A.B., M.G.S. 1 C: G.V., M.W., L.C., M.I.D.B., D.B., M.G.S. 2A-C: G.V., M.M., M.G.S. 3A: G.V., M.M., M.I.D.B., D.B. 3B: A.S., G.F., AB, MGS. The data that support the findings of this study are available from the corresponding author on reasonable request. Appendix S1 Supporting Information Appendix S2 Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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