Dissertations and theses : Doctoral thesis
Life sciences : Biochemistry, biophysics & molecular biology
Life sciences : Multidisciplinary, general & others
Human health sciences : Neurology
Systems Biomedicine
Uriarte Huarte, Oihane mailto [University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > >]
University of Luxembourg, ​​Luxembourg
Mittelbronn, Michel mailto
[en] Parkinson's disease ; Neuroinflammation ; Microglia ; Mouse models ; Transcriptomic profile ; Microglia heterogeneity
[en] Parkinson´s disease (PD) is the most common movement disorder caused by dopamine deficiency owing to a loss of dopaminergic neurons within the substantia nigra (SN). So far, there is no cure available, hence understanding the mechanisms by which dopaminergic neurons degenerate is essential for the development of future treatment strategies. Recently, a potential role of neuroinflammation, and especially the activation of microglial cells in PD was suggested, not being secondary to neuronal death, but rather primarily implicated in PD pathogenesis. Hence, we have ventured in to study neuroinflammation and microglia activation in the context of PD using in vivo and in vitro mouse models.

Firstly, we addressed microglial heterogeneity in the healthy nigrostriatal pathway, the primary circuit affected in PD. By using single-cell RNA sequencing, we have identified four different microglial immune subsets within the midbrain and the striatum. Notably, we were able to distinguish a microglial subset with an immune alerted phenotype, which was mainly composed of microglial cells from the midbrain. The transcriptomic identity of this subset resembled partially to the one of inflammatory microglia. Additionally, in situ morphological studies, such as 3D reconstruction, revealed that microglia located within the midbrain is less complex than microglia with a striatal origin.

Secondly, we studied the potential role of neuroinflammation and microglia in PD progression by using a PD-like mouse model of a-synuclein (a-syn) seeding and spreading. In this study, pre-formed fibrils (PFF) were injected into the mice striatum, and a combined neuropathological and transcriptomic analysis was performed at two time points that have distinct and increasing levels and distribution of a-syn pathology across different brain regions (13 and 90 days post-injection). Interestingly, neuropathological quantifications at 90 days post-injection uncovered that neuroinflammation and microglial reactivity are linked to neurodegeneration. However, pathology neither correlates with neurodegeneration nor with a-syn aggregation. Importantly, at 13 days post-injection, the transcriptomic analysis of the midbrain revealed the dysregulation of several inflammatory pathways and pointed to the overexpression of neurotoxic inflammatory mediators. Furthermore, at this time point, the presence of a-syn oligomers was detected in certain areas of the brain. Subsequently, we hypothesised that at early stages of PD pathogenesis, the presence of a-syn oligomeric forms induces a robust inflammatory response of microglia, which can be further associated with neurodegeneration.

Thirdly, to understand if a-syn oligomers are the main inducers of microglial activation, we examined further the microglial inflammatory response to other a-syn conformations, monomers and fibrils (PFF1 and PFF2). For that, BV2 and primary microglial cells were exposed to the a-syn moieties at different concentrations and incubations times. Electron microscopy depicted some heterogeneity across the synthetic a-syn fibrils, suggesting that PFF1 and PFF2 were composed by different structures. Then, microglial reactivity to a-syn monomers and fibrils was investigated by RT-PCR, and no specific response of microglia to a-syn was encountered. Also, only one of the a-syn fibrils, the PFF1, decreased microglial phagocytic activity and reduced the expression of Il1b by microglia after LPS stimulation.

Concomitant to the findings in the a-syn seeding and spreading model, we attempted to elucidate the molecular profile of microglia associated with neurodegeneration. In this particular study, RNA-sequencing was performed in isolated microglial cells in an early stage of pathology progression. In contrast with our previous results, no differences in the microglial profile were found between the PFF and the control mice.

Lastly, we have investigated potential neuroprotective mechanisms associated with microglial reactivity counter-regulation. Considering previous observations that microglia express dopaminergic receptors, we investigated further whether apomorphine, a dopamine agonist with anti-oxidant properties, could govern microglial activation. The effect of apomorphine enantiomers was analysed within primary microglia cultures that were activated by exposure to mutated A53T monomeric a-syn. Herein, we demonstrated that microglial activation can be dampened by apomorphine, via the recruitment of Nrf2 to the nucleus, which results in a decreased release of proinflammatory mediators, such as TNFa or PGE2.

Taken together, this study provides an additional characterisation of neuroinflammation and microglial cells in the context of PD, which ultimately contributes to a better understanding of their relationship with neurodegeneration.
Luxembourg Centre for Systems Biomedicine (LCSB): Luxembourg Center of Neuropathology (Mittelbronn group)

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