In this study, we aimed to design a method based on the fluorescence NTA of the salivary sEV to address the benefits of early diagnosis of PD. The role of sEV in the propagation of disease pathologies in neurodegenerative diseases and psychiatric diseases is well known [
10,
17,
31,
32]. The specific trigger due to which the monomeric form of α-syn acquires the neurotoxic oligomeric form and makes the large aggregates that result in Lewy pathology is due to disturbances in the lysosomal autophagy system (LAS) that leads to inefficient clearance of α-syn oligomeric assemblies [
33,
34]. The increased sEV secretion and the transfer of disease pathologies make them a potential candidate that can give a fingerprint of the molecular status of their originating cell. Our study attempts to explicate the fluorescence-tagged salivary sEV 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 sEVs concentration in PD patients in all modes of NTA. The sEV were isolated using a rigorous isolation protocol combining the chemical precipitation method followed by ultrafiltration [
30]. The sEV suspension from precipitation is followed by an ultrafiltration step that aids in concentrating the sEV suspension and removal of traces of precipitant and very small protein contaminants. We identified the purity and efficient isolation of sEV using CD63 (surface marker) expression in the sEV pellet and supernatant. We also used the neuronal protein L1CAM to check the neuronal origin, although the use of this marker for specific CNS-derived vesicles is contradicted [
35]. Nevertheless, in our study, the neuron-related protein is used to check the protein markers and not for L1CAM affinity-based isolation. Two steps filtration procedure has accompanied the sEV isolation method in our study to ensure high purity.
Nonetheless, with the fluorescent-dye-labeled salivary sEVs, we achieved the sensitivity and specificity (AUC = 0.967, 94.34% sensitivity, 90.91% specificity) that can be clinically acceptable for a method. This fluorescent dye specifically binds to the lipid bilayer of the plasma membrane. Thus, distinctively it binds to the sEV in the nanometer range and effectively distinguishes them. To validate the fluorescence-tagged results, we worked on an Alexa fluor 488 conjugated anti-CD63 antibody on sEV. The CD63-antibody-labeled salivary sEV concentration supported the fluorescence-tagged results with similar accuracy (AUC of 0.9191, a sensitivity of 94.12%). sEVs have a heterogeneous population of the protein, and hence, not all sEVs carry similar markers; instead, the markers are changing depending on the sEV subset [
35,
36]. Therefore, to characterize sEVs, different markers can be used to observe the overall population of sEVs [
10,
30,
36]. To reduce the variations between results, we used different modes of NTA to characterize the heterogeneous population of sEVs in saliva samples from controls, prodromal, and PD. To further substantiate our findings, we evaluated the expression profiles of sEV markers (CD9, CD63, Flotillin-1), a neuronal marker (L1CAM), and PD specific phospho-α-synuclein in isolated sEVs from PD patients and healthy controls. In this study, PD patients show significantly increased expressions of sEV markers (CD9,
p = 0.0004; CD63,
p = 0.0017; Flotillin-1,
p = 0.0213), a neuronal marker (L1CAM,
p = 0.0253) and PD specific phospho-α-synuclein (
p = 0.0093) than healthy controls due to higher numbers of sEV in PD patients as observed in the NTA experiments. It is also reflected in similar trends in loading control as housekeeping protein expression should also change with the number of sEV. Similarly, the differential expression of α-syn from the salivary sEV cargo detected by ELISA is significantly higher in PD than in HC.
The ROC curve analysis for the α-syn expression has an AUC of 0.8137 and a sensitivity of 88.24%. Our study's primary outcomes contradicted the total α-syn levels in the CSF [
37,
38] but aligned with plasma and saliva α-syn levels [
39] detected by immunoassays in the previously published studies. Nonetheless, the data from the meta-analysis of total CSF α-syn shows low diagnostic accuracy [
37], whereas our approach resulted in a similar AUC of 0.96 in the case of the fluorescent dye-labeled salivary sEV. The outcome of our validation study on sEV cargo, α-syn
Total showed 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%) [
38]. In establishing the correlation between the PD hallmark protein α-syn and the fluorescent dye-labeled salivary sEV, we observed a positive correlation of
r = 0.4844 and a significant
p-value (0.0416). Furthermore, we propose for the first time how the fluorescent-dye-labeled salivary sEV and α-syn are correlated. A study published by Cao et al.(2019) shows the level of salivary sEV α-syn
Olig and α-syn
Olig/α-syn
Total in HC versus PD Western blot profiling and obtained the AUC = 0.941, 92% sensitivity, 86% specificity for α- syn
Olig as well the AUC = 0.772, 81% sensitivity and 71% specificity for α-syn
Olig/α-syn
Total [
40], whereas our study independently with the fluorescent-dye-labeled salivary sEV supported with the antibody (α-syn
Total) has higher diagnostic accuracy. Although the antibody-based determination of sEV concentration is more specific to sEV-surface markers; however, it is expensive, and chances of variations occur due to several steps, whereas the fluorescence dye-based method is easy and cost-effective, henceforth proving to be more suitable for developing a possible robust detection protocol. Our group is also working on other neurological disorders, i.e., Alzheimer’s disease (AD), where we observed the changes in the sEV concentration in control, mild cognitive impairment (MCI), and AD [
25]. For the confirmed diagnosis, the anatomical imaging (CT, MRI) does not show significant differences between PD and other PD-like conditions; therefore, the approaches like positron emission tomography (PET) and SPECT with the radio-labeled molecules that specifically bind with the target are found to be more efficient [
41‐
44].
99mTc-TRODAT-SPECT/CT, in which the Technetium-99 mm binds with the dopamine transporter (DAT), is target-specific and serves as a representation of the density of dopaminergic neurons [
45,
46]. In our study, we quantified the binding of Technetium-99 m to the DAT. The uptake ratios in the bilateral whole striatum, caudate, and putamen are higher in the healthy age-matched controls compared to PD, thus confirming the diagnosis of the patients. The differences between the binding ratios of HC and PD are mentioned in Table
3. Some of the earlier studies suggested the correlation between the striatal ratios and the α-syn
Total in the blood plasma [
47] of the PD, and our results displaying the positive trend of the striatal ratios and α-syn
Total are in concordance with it.