• BIOMARKERS
• NEURODEGENERATION
• NEUROINFLAMMATION
• PARKINSON

Parkinson's disease (PD) is a neurodegenerative disorder caused by the progressive loss of dopaminergic neurons located in the substantia nigra pars compacta of the midbrain. It is known that the degeneration of neurons in PD is associated with the formation of intracellular alpha-synuclein aggregates that are extremely toxic for cells and contribute to the neuroinflammatory response, nevertheless the promoting causes and the precise mechanism that leads to neurodegeneration have to be fully elucidated. Some of the cytokines such as IL6, α-synuclein and regulatory miRNAs released by immune cells into blood may contribute to disease progression and represent useful biomarkers for Parkinson’s disease.
The aim of this thesis is to verify the role of inflammation and to search biomarkers related to disease onset in blood samples of PD patients. We have focused our attention on inflammation and/or viral infections related with the risk of developing PD.
We have enrolled a group of 65 PD patients, over 50 years old, which undergo periodic medical examinations and blood withdrawal at the Neuro-Rehabilitation Center of the Azienda Ospedaliera Universitaria Pisana. The research of inflammation related biomarkers was performed using blood samples processed to obtain plasma, serum and leucocytes of the most representative PD patients.
It has been reported that lymphocytes, the main cell type of the immune system, are a peripheral model of CNS neurons, because they infiltrate through the Blood Brain Barrier and establish a sort of crosstalk with neurons. Moreover, it has been suggested that innate immune system possibly contribute to neurodegeneration, therefore, leucocytes could represent an important source of biomarkers. We selected leucocytes from blood samples and assayed the Syntase 1 expression, a protein activated by Interferon beta increase during inflammation. Our preliminary results show that Syntase 1 is expressed and induced into leucocytes of PD. The interferon beta activation seems to be related with the miRNA 155, one of the main miRNAs involved in regulating immune response, viral infection and α-synuclein in the CNS. In case of viral infection miRNA 155 promotes the activation of the adaptive immune system regulating T lymphocytes and Natural killer cells proliferation, as well as, in presence of α-synuclein aggregates, miRNA 155 promotes the activation of the immune response increasing the deleterious effects of immune reactivity on neurodegeneration. We have investigated miRNA 155 expression into plasma and leucocytes, correlating its levels with the expression of the α-synuclein. Preliminary results have shown that miRNA 155 is differentially expressed into leucocytes of controls respect to patients.
Since exosomes are the main carrier of miRNA into blood and the vehicles of cellular information during pathologies, we have extracted exosomes from plasma by Size Exclusion Chromatography (SEC), characterized their nucleic acid, protein content and examined their morphology by Transmission Electron Microscope and Nanosize equipment. We assayed the IL-6 levels by ELISA test and Mass Spectrometry on total exosomes to identify transported proteins. The IL6 level resulted altered in PD patients. Mass Spectrometry indicated that total exosome preparations were contaminated by plasmatic proteins, so this convinced us to use a procedure to enrich samples in neuronal derived exosomes. The enrichment was performed by applying an immunoplate coated with specific neuronal exosomal antibodies that allowed us to obtain an increase in neuronal exosomes of more than 50%.
We developed a database including symptom onset, clinical data, progression rate, together with 108 different blood analytes collected during periodical visits, for each patient across the disease course, to perform a statistical analysis on blood analytes. Clinical data included demographics, medical history, treatments, and disease severity. A statistical analysis using machine-learning procedure was performed to identify a correlation between blood analytes associated with neuroinflammation and disease severity. Results were compared to miRNA and mRNA content of PD patients examined and with other neurodegenerative diseases such as Alzheimer Disease or Amyotrophic Lateral Sclerosis.
The use of blood circulating biomarkers would be useful tools for the diagnosis of Parkinson disease, before dopaminergic neurons undergo complete degeneration and all motor symptoms are worsened. This plan could be performed with particular advantages as the collection of the sample is not invasive and the detection of miRNA and mRNA does not require complex techniques.