Patricia Valerio Santos

Background: The identification of circulating biomarkers that closely correlate with Parkinson's Disease (PD) has failed several times in the past. Nevertheless, in this pilot study, a translational approach was conducted, allowing the evaluation of the plasma levels of two mitochondrial-related proteins, whose combination leads to a robust model with potential diagnostic value to discriminate the PD patients from matched controls. Methods: The proposed translational approach was initiated by the analysis of secretomes from cells cultured under control or well-defined oxidative stress conditions, followed by the identification of proteins related to PD pathologic mechanisms that were altered between the two states. This pipeline was further translated into the analysis of undepleted plasma samples from 28 control and 31 PD patients. Results: From the secretome analysis, several mitochondria-related proteins were found to be differentially released between control and stress conditions and to be able to distinguish the two secretomes. Similarly, two mitochondrial-related proteins were found to be significantly changed in a PD cohort compared to matched controls. Moreover, a linear discriminant model with potential diagnostic value to discriminate PD patients was obtained using the combination of these two proteins. Both proteins are associated with apoptotic mitochondrial changes, which may correspond to potential indicators of cell death. Moreover, one of these proteins, the VPS35 protein, was reported in plasma for the first time, and its quantification was only possible due to its previous identification in the secretome analysis. Conclusions: In this work, an adaptation of a translational pipeline for biomarker selection was presented and transposed to neurological diseases, in the present case Parkinson's Disease. The novelty and success of this pilot study may arise from the combination of: i) a translational research pipeline, where plasma samples are interrogated using knowledge previously obtained from the evaluation of cells' secretome under oxidative stress; ii) the combined used of statistical analysis and an informed selection of candidates based on their link with relevant disease mechanisms, and iii) the use of SWATH-MS, an untargeted MS method that allows a complete record of the analyzed samples and a targeted data extraction of the quantitative values of proteins previously identified.

Parkinson’s disease is the second most common neurodegenerative disease, characterized by four motor symptoms: rest tremor, bradykinesia, rigidity and postural instability. Diagnosis is made through strict clinical criteria, but clinical diagnosis is difficult in the early disease state and frequently does not allow a definitive diagnose. One hallmark of Parkinson’s disease is the death of the pigmented dopaminergic neurons of midbrain substantia nigra, with consequent loss of the neurotransmitter dopamine in the corpus striatum. The exact mechanisms underlying are still not understood and current theories include neuroinflammation and oxidative stress. Evidences suggest the presence of chronically activated microglia and several studies detected higher levels of their markers, namely cytokines and oxidative stress products. The main objectives of this study were to characterize CHCB’s Parkinson population, to evaluate inflammatory and oxidative markers and immunological status. Medical records were evaluated for patients (n=38) and healthy controls (n=32). Patient’s motor symptoms at onset and at evaluation were determined. The inflammatory markers measured were C reactive protein, erythrocyte sedimentation rate and serum cytokines (interleukins 1ß, 8, 6, 10, 12p70 and Tumor Necrosis Factor). Immunological status was accessed through leukogram. Urate was our oxidative marker. The cytokines measurement reveals values below the lower limits of detection for all. C reactive protein was higher in patients, but erythrocyte sedimentation rate was similar between both groups. In leukogram analysis, we detected higher values of neutrophils in patients. However, lymphocytes were the only that decrease throughout the temporal evolution of the disease and in patients using higher doses of L-DOPA. We attempted to clarify the real cause of lymphocytes decrease. Although there was a tendency to observe a lower number of lymphocytes for higher doses of L-DOPA in all phases of temporal evolution of the disease, the difference was not statistically significant. In relation to urate, we obtained lower values in patients and the decrease was marked in the higher stages of the disease and longer disease duration. Regarding inflammatory aspects of the disease, our study showed that cytokine pattern is not sensitive or specific to assess disease progression. On the other hand, the antioxidant urate presents us as a putative marker of disease severity.

Additional file 6: Supplementary Table 4. Evaluation of the total levels of the plasma proteins of Controls and Parkinson’s disease patients.

Additional file 5: Supplementary Table 3. Evaluation of the total levels of the secreted proteins under control and stress conditions.