Plasma extracellular vesicle synaptic proteins as biomarkers of clinical progression in patients with Parkinson’s disease: A follow-up study

Reviewer #1 (Public Review):

The study isolated extracellular vesicles (EV) from healthy controls (HCs) and Parkinson patients (PwP), using plasma from the venous blood of non-fasting people. Such EVs were characterized and validated by the presence of markers, their size, and their morphology. The main aim of the manuscript is to correlate the presence of synaptic proteins, namely SNAP-25, GAP-43, and SYNAPTOTAGMIN-1, normalized with HSP70, with the clinical progression of PwP. Changes in synaptic proteins have been documented in the CSF of Alzheimer's and Parkinson's patients. The demographics of participants are adequately presented. One important limiting, as well as puzzling aspect, is the fact that authors did not find differences between groups at the beginning of the study nor after one year, after age and sex adjustment. Tables in general are hard to follow. Specifically, Table 2 does not convey a clear message nor in the text of the Table itself, and the per 100% of change needs to be explained in the corresponding legend. It is only when PwP were classified as a first quartile that a significantly greater deterioration was found. However, in the case of tremor, the top 25% had values going from 0.46-0.47 to 0.32-0.35, whereas the lower three quarters went from 0.33-0.34 to 0.27-0.28 depending on the protein analyzed. This needs to be clarified in the text. Table 3 is hard to read and some of the values seem repetitive, especially for tremor, AR, and PIGD. It looks as if Figure 2 represents the same information as Table 3. The text and figure legends are not helpful in guiding the reader to understand the presented information.

Reviewer #2 (Public Review):

Hong and collaborators investigated variations in the amount of synaptic proteins in plasma extracellular vesicles (EV) in Parkinson's Disease (PD) patients on one-year follow-up. Their findings suggest that plasma EV synaptic proteins may be used as clinical biomarkers of PD progression.

It is a preliminary study using semi-quantitative analysis of synaptic proteins.

The authors have a cohort of PD patients with clinical examination and a know-how on EV purification. Regarding this latter part, they may improve their description of EV purification. EV may be broken into smaller size EV after freezing. Does it explain the relatively small size in their EV preparation? Do the authors refer to the MISEV guidelines for EV purity? Regarding synaptic protein quantification, the choice of western blotting may not be the best one. ELISA and other multiplex arrays are available. How the authors do justify their choice? Do the authors try to sort plasma EV by membrane-associated neuronal EV markers using either vesicle sorting or immunoprecipitation?

Many technical aspects may be improved. Such technical questions weakened the authors' conclusions.

The discussion is pretty long to justify the data. It may be shortened by adding some information in the introduction.