Di Martino, Rossana (2019) Role of mitochondria in the activation of neuroinflammation in animal models of alpha-synucleinopathies. [Tesi di dottorato]

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Abstract

The purpose of the present study was to investigate the possible role of the mitochondria in the onset of neuroinflammation in vitro and in vivo models of PD. In particular, it was investigated the relationship between the dysregulation of mitochondrial calcium content and mitochondrial dysfunction and its consequences on the activation of the neuroinflammatory process. To address this issue experiments were performed in animal models of familial forms of PD, such as mice carrying the human mutation of α-synuclein A53T under the Prion murine promoter, and mice expressing wild-type human -synuclein under the regulatory control of the platelet-derived growth factor-β (PDGF-β) promoter. The first part of this study was performed in vitro, in primary neuronal and glial cells obtained from midbrain and striatum of A53T transgenic and wild type mice. In this model, mitochondrial calcium content and membrane potential were measured simultaneously with intracellular calcium concentration by using confocal microscopy and fluorescent dyes. Furthermore, Western blot experiments were performed ex vivo in midbrain and striatum obtained from A53T and WT mice during ageing to evaluate mitochondrial dysfunction and neuroinflammation in A53T transgenic in comparison with WT. To this aim the expression of Cytochrome-c (Cyt-c), a marker of mitochondrial damage, and Manganese superoxide dismutase (MnSOD) and Neuronal Nitric oxide synthases (nNOS) two markers of mitochondrial oxidative stress, were measured in 4-12- and 16- month- old mice. Finally, to correlate mitochondrial dysfunction to neuroinflammation, further experiments were performed ex vivo in 4- 12- and 16-month- old A53T and WT mice with the aim to evaluate the expression levels of pro-inflammatory proteins such as the inducible Nitric oxide synthases (iNOS) and Interleukin 1 beta(IL-1β). The last part of the study was addressed to delve deeper into the understanding of the role played by mitochondrial dysfunction in the pathogenesis of neuroinflammation observed in PD. To this aim experiments were performed in collaboration with the “Deutsches Zentrum fΰr Neurodegenerative Erkrankungen” (Centre for Neurodegenerative Disease), with the intent to study the proteomic profile of microglial cells isolated from PDGF-h-α-synuclein mice 2 and 12 months old, and to correlate the changes in mitochondrial protein expression with the onset of the neuroinflammatory process leading to neurodegeneration occurring in PD. To further confirm the role played by glial cells in the pathogenesis of neuroinflammation leading to neuronal dysfunction observed in PD progression, experiments were performed in vitro in astrocytes obtained from A53T transgenic and WT mice, in which NCX1 expression and mitochondrial function were assessed by western blot analysis and confocal microscopy.

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