In this study, we aimed at designing a method based on the fluorescence NTA of the salivary exosomes to address the benefits for early diagnosis of PD. The role of exosomes in the propagation of disease pathologies in neurodegenerative diseases and psychiatric diseases is well known13,20,27,28. Specifically, in PD the prion-like propagation of misfolded- α-syn via exosomes and how the exosomes can seed the intraneuronal-α-syn to other regions of the brain, has become a prospective mechanism of spread of α-syn pathology. The disturbances in the lysosomal autophagy system (LAS) and the ubiquitin proteasomal system are some of the reasons for aggregation of the misfolded α-syn. The specific trigger due to which the monomeric form of α-syn acquires the neurotoxic soluble oligomeric form and makes the large aggregates which result in Lewy pathology is mostly due to the disturbances in the α-syn degradation pathway. The LAS has already established its role as a premier pathway for the clearance of α-syn oligomeric assemblies29,30. The increased exosomes secretion as well the transfer of disease pathologies makes them a potential candidate that can give a fingerprint of the molecular status of their originating cell. Our study is an attempt to explicate the fluorescence tagged salivary exosomes that has similar relationships with the hallmark protein (α-syn) for the diseases and this can be used as an early diagnostic methodology in PD.
The key findings of this study revealed the increased salivary exosome concentration in PD patients in all modes of NTA, which is indicated in the earlier reported outcomes21–23,25. The isolated exosomes from the saliva samples of subjects are characterised and validated using the MISEV 2018 criteria established to work with specifically with extracellular vesicles31. We have used protein-based extracellular vesicle characterisation criteria to assess the purity of the methods of exosome isolation and demonstrated efiicient separation from other extracellular vesicles. The protein markers from categoeries 1,2, and 3 are used in analysis. Apart from this we have used the neuronal protein L1CAM to check the neuronal origin, although the use of this marker for specific CNS derived vesicles is contradicted. Nevertheless, the literature in the area of extracellular vesicels and the use of L1CAM is scarce and in our study the neuron-related protein is only used to check the protein markers not for L1CAM affinity based isolation. The elusiveness encircling around L1CAM calls for more studies although not denying the association of L1CAM with extracellular vesicles. The exosome isolation method used in our study, we have accompanied it with the two steps filtration procedure to ensure high purity. Mean concentration of exosomes all modes of NTA was found to be increased in PD in comparison to HC. However, with the fluorescent dye-labeled salivary exosomes, we achieved the sensitivity and specificity that can be clinically acceptable for a method. The concentration differences between PD and HC observed an AUC of 0.967, a sensitivity of 94.34% and, a specificity of 90.91%. This fluorescent dye, used for the experimentation specifically binds to the lipid bilayer of the plasma membrane. Thus, distinctively it binds to the exosomes in the nanometer range and effectively distinguishes them. To validate the fluorescence tagged results, we worked on a CD63 primary antibody conjugated with Alexa fluor-488 that binds particularly to the tetraspanin CD63 on exosomes in NTA mode. The CD63-antibody labeled salivary exosomes concentration supported the fluorescence tagged results with similar accuracy (AUC of 0.9191, sensitivity of 94.12%). The differential expression of α-syn (hallmark for PD disease pathology) from the salivary exosomes cargo detected by ELISA is found to be significantly higher in PD cases when compared to HC. The ROC curve analysis for the α-syn protein has an AUC of 0.8137, and sensitivity of 88.24%.The preceeding outcomes of our study were in contradiction with the total α-syn levels in the CSF32,33 but aligned with plasma and saliva α-syn levels34 detected by immunoassays in the previously published studies. Nonetheless, the data from the meta-analysis of total CSF α-syn shows low diagnostic accuracy33, whereas our approach had resulted in a similar AUC of 0.96 in the case of the fluorescent dye-labeled salivary exosomes. The outcome of our validation study on exosomal α-syn total, showing a better sensitivity and specificity (AUC: 0.81, sensitivity: 88%, specificity: 75%) in comparison to the other work (AUC: 0.657 sensitivity:71.2%, specificity 50.0%)32. On, establishing the correlation between the PD hallmark protein α-syn and the fluorescent dye-labeled salivary exosomes, we observed a positive correlation r = 0.4709 and a significant p-value (0.0486). In this study, we have proposed for the first time how the fluorescent dye-labeled salivary exosomes and α-syn are correlated. A study published by Cao et al.(2019) shows the level of salivary small extracellular vesicular cargo α-synlig & α-synOlig/α-synTotal in HC versus PD western blot profiling and obtained the AUC of 0.941, the sensitivity of 92% and specificity 86% for α-synOlig as well the AUC of 0.772, sensitivity 81% and specificity 71% for α-synOlig/α-synTotal35, whereas our study independently with the fluorescent dye-labeled salivary exosomes supported with the antibody (α-synTotal) has higher diagnostic accuracy. Although the antibody-based determination of exosome concentration being more specific to exosome-surface markers seemed highly appropriate for the development of methodology. But the antibody-based diagnostic method is expensive and variations occur due to involvement of several steps, whereas the fluorescence dye-based method is easy and cost-effective henceforth proving to be more suitable for developing a possible robust screening protocol.
PD-like symptoms are seen in many conditions other than the idiopathic PD, like multiple system atrophy (MSA), progressive supranuclear palsy (PSP), corticobasal-degeneration (CBD). For the confirmed diagnosis, apart from the clinical profiling molecular medicine imaging has always shown high potential in comparison to anatomical imaging like MRI (magnetic resonance imaging) and CT (computed tomography). The anatomical imaging doesn’t show highly significant differences between the PD and other PD-like conditions. Therefore, the approaches like Positron emission tomography (PET) and single-photon emission computed tomography with the radio-labeled molecules that specifically bind with the target are found to be more efficient36–39. 99mTc-TRODAT-SPECT/CT, in which the Technitium 99mm binds with the dopamine transporter (DAT), a membrane protein at the pre-synaptic terminal of dopaminergic neurons, is target-specific. The binding at the DAT serves as a representation of the density of dopaminergic neurons40,41. In our study, we have given the quantification of the binding of Technitium 99m to the DAT. The uptake ratios in the bilateral whole striatum, caudate, and putamen with occipital cortex as a background are calculated. The uptake ratios are higher in the healthy age-matched controls in comparison to PD, thus confirming the diagnosis of the patients. The differences between the binding ratios of HC and PD are mentioned in Table 3. The difference in the binding ratios between the subjects depends upon the activity of the DAT in the region of interest. Some of the earlier studies suggested the correlation between the striatal ratios and the α-synTotal in the blood plasma42 of the PD and our results displaying the positive trend of the striatal ratios & α-synTotal are in concordance with it.
In the process of our analysis, we found some of the recruited healthy control samples had higher concentrations of these, fluorescent-tagged salivary exosomes, and α-synTotal in comparison to HC and the lower concentrations in the comparison of the PD samples. We assumed these subjects might be in the asymptomatic stages of Parkinson’s disease, although confirmed diagnosis is not possible as they were not subjected to TRODAT scans. Nevertheless, this can show that the fluorescent-tagged salivary exosomess could be a molecular signature that has the potential to act as a susceptibility-risk biomarker.