Análise do perfil proteômico de exossomos enriquecidos do plasma sanguíneo de pacientes com Doença de Parkinson

Abstract

Parkinson’s disease (PD) is the second most common neurodegenerative disease, affecting millions of people worldwide. So far, there is no biomarker in the blood to identify the disease and the diagnosis is exclusive clinical. PD is clinically characterized by four main symptoms: bradykinesia, tremor at rest, postural instability and stiffness, being characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta and formation of alpha-synuclein aggregates, called Lewy bodies. Regarding progression, PD can be classified according to the Hoehn and Yahr staging scale where patients are classified as mild, moderate or advanced according to the disease-related motor impairment. Exosome studies indicate a mediation intercellular communication through direct cell-cell contact or through the transfer of secreted molecules. Exosomes are nanovesicles (40-120 nm) surrounded by a Lipid bilayer that can contain nucleic acids, metabolites, specific proteins, among other insides. Although many efforts have been made to reveal the mechanisms involved in the pathogenesis of PD, there is still much to discovered, especially regarding diagnostic and prognostic molecular biomarkers. The general objective of this work was to analyse the proteomic profile of blood plasma enriched exosomes of patients with Parkinson’s disease. For this, blood samples were collected in collaboration with the neurology department of the UFRJ University Hospital. A cohort of 32 plasma samples from healthy controls and 97 samples from PD patients clinically classified as mild (n=44), moderate (n=41) and advanced (n=12) were used. Four pools were set up, which had the exosomes enriched using the ExoQuick® Kit. From these exosomes, protein extraction and subsequent shotgun proteomic analysis were performed; LC- MS/MS (Q-Exactive plus, EASY-nLCII, Thermo Fisher Scientific) of these samples and the results were analyzed by bioinformatics. A total of 671 proteins were identified redundantly in the sum of samples from patients with PD and 229 proteins in the healthy control group. In a non-redundant way, a total of 260 proteins were identified in all groups in the present study. 8, 9, 7, 9, proteins were identified exclusively present in the mild, moderate, advanced and control group, respectively. Of the total number of non-redundant proteins, 234 met the criteria of the PERSEUS software and went on to quantitative analysis. When comparing the mild versus control group, a total of 42 differentially expressed proteins were identified. When comparing the moderate versus control groups, a total of 33 differentially expressed proteins were identified. And in the comparison of the advanced versus control group, a total of 16 differentially expressed proteins were identified. Pathways related to platelet activation, activation of the complement system and its cascade and pathways of the innate immune system were identified. In addition, significantly increased levels of hydroperoxide were identified in the exosomes of the group with mild clinical classification in relation to the control group and the group with moderate clinical classification. The antioxidant level was measured and significant depletion was observed in all clinical groups when compared to the control group. The oxidative stress index showed a significantly high level in the groups with mild and moderate clinical classification in relation to the control group.