Single molecule characterisation of salivary protein aggregates from Parkinson's disease patients – a pilot study

Saliva is a convenient and accessible biofluid that has potential as a future diagnostic tool for Parkinson’s disease. Candidate diagnostic tests for Parkinson’s disease to date have predominantly focused on measurements of alpha-synuclein in cerebrospinal fluid, but there is a need for accurate tests utilising more easily accessible sample types. Prior studies utilising saliva have used bulk measurements of salivary α-synuclein to provide diagnostic insight. Aggregate structure may influence the contribution of α-synuclein to disease pathology. Single molecule approaches can characterise the structure of individual aggregates present in the biofluid and may therefore provide greater insight than bulk measurements. We have employed an antibody-based single-molecule pulldown assay to quantify salivary α-synuclein and amyloid-β peptide aggregate number and subsequently super-resolved captured aggregates using direct Stochastic Optical Reconstruction Microscopy to describe their morphological features. We show that the salivary α-synuclein aggregate/amyloid-β aggregate ratio is increased almost two-fold in Parkinson’s disease patients (n = 20) compared to controls (n = 20, p <0.05). Morphological information also provides insight with saliva from Parkinson’s disease patients containing a greater proportion of larger and more fibrillar amyloid-β aggregates than control saliva (p <0.05). Furthermore, the combination of count and morphology data provides greater diagnostic value than either measure alone distinguishing between the people with Parkinson’s disease (n = 17) and controls (n = 18) with a high degree of accuracy (AUC = 0.87, p <0.001) and a larger dynamic range. We therefore demonstrate for the first time the application of highly sensitive single molecule imaging techniques to saliva. In addition, we show that aggregates present within saliva retain relevant structural information further expanding the potential utility of saliva-based diagnostic methods.