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|Authors: ||Alberio, Tiziana|
|Internal Tutor: ||FASANO, MAURO|
|Title: ||The proteomic approach to investigate Parkinson's desease: pathogenetic mechanisms and biomarkers discovery.|
|Abstract: ||The overall goals of basic research in improving the clinical approach to a disease are, on the one hand, to unravel its natural history, finding its cause and developing better treatments and, on the other hand, to find biomarkers that would permit to ameliorate the diagnosis, to distinguish among patient subtypes and to set up clinical trials of new therapeutic agents. The common threads of the two projects developed in this thesis are the technique used, proteomics, and the main object, Parkinson‟s disease (PD). Proteomics is a powerful methodology to investigate how protein expression is affected in the pathogenesis of a disease process and should be exploited both to investigate PD pathogenetic mechanisms and to discover peripheral biomarkers.
In the first part of this thesis the project “A SH-SY5Y model to investigate PD pathogenetic mechanisms” is discussed. We investigated the expression pattern of cellular proteins following dopamine exposure in catecholaminergic SH-SY5Y human neuroblastoma cells overexpressing α-synuclein by a proteomic approach. Using 2D gel image analysis and ANOVA statistics we identified proteins that correlate with the experimental conditions in our model. Significant changes in specific cellular processes were observed, such as cytoskeleton structure and regulation, mitochondrial function, energetic metabolism, protein synthesis and neuronal plasticity. Proteins were analyzed by a network enrichment tool that automatically enriched the network model in terms of the most relevant proteins that seem to be missing. Moreover, the bioinformatic approach found out the enriched Gene Ontology (GO) categories, allowing us to draw attention to the NF-κB pathway. To validate experimentally the hypothesis of the NF-κB involvement, we performed a luciferase gene reporter assay that revealed a downregulation of the transcription factor activation by α-synuclein increased expression and its complete quenching by dopamine exposure.
Each element arising from the present study could represent a valuable starting point for focused investigations aimed to better understand key issue of PD pathogenesis. For instance, a protein that completely disappeared after dopamine treatment was VDAC2, a porin of the outer mitochondrial membrane. Since the impairment of mitochondrial function is considered among the major pathogenetic factors of neurodegeneration in PD, we further investigated the regulation of VDAC2 degradation and the role of its phosphorylation.
Accumulating evidence supports a specific role of DJ-1 in protecting dopaminergic neurons from dopamine itself. Since this protein act as a pool of different forms possibly associated to different function and localization, we analyzed DJ-1 2D electrophoresis pattern in SH-SY5Y cells after exposure to dopamine; we observed a specific increase in the most acidic forms in the pattern together with a significant decrease of the most basic spot. Unlike cells exposed to generic oxidative conditions, no additional shift was observed. The results are corroborated by a meta-analysis of the literature showing that in the absence of dopamine treatment the specific acidic form is underrepresented.
The second part of the thesis is focused on the project “Dopamine response of dopaminergic circulating cells: the Jurkat cell model”. The functions of immune cells are regulated not only by cytokines, but also by several neurotransmitters. In particular, peripheral blood lymphocytes possess a complex dopaminergic regulatory system. Human T-lymphocytes express all five dopamine receptors (D1-D5), each of which exerts different actions on the regulation of T-cell functions. The Jurkat human leukemia CD4+ T-cell line has been thoroughly used and characterized as a suitable cell model to investigate T-cell signaling and apoptosis, nevertheless its characterization as a model of circulating dopaminergic cells was never reported before. In this regard, we characterized the dopaminergic system in Jurkat cells and through a proteomic approach we analyzed their response to a dopamine challenge in order to highlight metabolic pathways that could specifically reveal alterations linked to PD. A dopamine challenge, with no effect on cell viability (50 μM), induced quantitative changes in the protein expression pattern. The proteins that showed quantitative differences were identified by peptide mass fingerprinting and analyzed in terms of both interaction network and GO classification enrichment. We obtained a significant model for network enrichment and a functional association to GO classification (unfolded protein binding). Eventually, we observed a modification of β-actin and 14-3-3 two-dimensional pattern following dopamine exposure.
In both projects, by combining the experimental and the bioinformatic approach, we fulfilled the expectations for proteomics to generate new hypotheses. Of course, one must consider that models have limitations and that hypotheses need further validation, but they could be a good starting-point to unravel biochemical pathways altered in PD, a disease with a poorly understood etiology.|
|Subject MIUR : ||BIO/10 BIOCHIMICA|
|Issue Date: ||2010|
|Doctoral course: ||Neurobiologia|
|Academic cycle: ||23|
|Publisher: ||Università degli Studi dell'Insubria|
|Citation: ||Alberio, T.The proteomic approach to investigate Parkinson's desease: pathogenetic mechanisms and biomarkers discovery. (Doctoral Thesis, Università degli Studi dell'Insubria, 2010).|
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