217 Chapter 3 expands on the previous chapter and features an in-depth characterisation of TSPO expression in MS lesions using multiple markers of microglia (IBA1, HLA-DR, and CD68). We also investigated whether TSPO+ homeostatic microglia co-expressed markers of activation. Additionally, we investigated the amount of TSPO expressed by microglia in MS lesions and correlated this with morphological data. In this study, we replicated findings of chapter 2 but also found that almost all TSPO+ cells across a range of white matter regions were microglia, and that the phenotype in control and NAWM was homeostatic, and the phenotype in active and the rim of chronic active lesions was activated. More importantly we found no differences in the expression of TSPO in activated microglia when compared to microglia that were homeostatic in NAWM and control white matter nor did we find differences in morphology that indicated changes in TSPO expression. Overall, this indicates that TSPO PET could best be interpreted as a readout for total microglia count instead of the activation states of local microglia. Chapter 4 investigates TSPO expression in glial cells in multiple neurodegenerative diseases, namely AD, ALS and MS and how TSPO is expressed in their commonly used rodent models. Sincemany studies have assumed TSPOPET to reflect an increase in TSPO in activatedmicroglia we tested whether this assumption was holding true for these neurodegenerative diseases and rodent models. We performed a meta-analysis of publicly available expression array data and found that across a range of pro-inflammatory activation stimuli, TSPO expression is consistently and substantially increase in mouse, but not human macrophages and microglia in vitro. Afterwards we performed a comparative analysis of the TSPO promotor region in a range of mammalian species and found that the binding site for AP1 (a transcription factor which regulates macrophage activation) is present in and unique to a subset of species within the Muroidea superfamily of rodents and absent in humans. Consistent with the in vitro data we found that postmortem microglia in AD, ALS and MS, and in marmoset EAE do not increase TSPO expression when in a pro-inflammatory state. Contrastingly, TSPO expression is markedly increased in activated myeloid cells in all mouse models of these diseases. Lastly, we investigated relative expression of TSPO in publicly available single cell RNA sequencing datasets of the CNS of AD and MS and their respective animal models. Again we found no evidence for an increase of TSPO expression in the human CNS disease in activated microglia while mouse microglia did show an increase in Tspo expression. These data suggest that TSPO PET is sensitive to microglial activation is true only for a subset of species within the Muroidea superfamily of rodents. In contrast, in humans and other mammals, it simply reflects the local density of inflammatory cells irrespective of the disease context. The clinical interpretation of the TSPO PET signal therefore needs to be revised. Chapter 5 summarises all the published studies on TSPO expression in the CNS in healthy and diseased brains. We provide an overview of TSPO expression in commonly used rodent models and in common human neurodegenerative and neuropsychiatric diseases. Based on the literature we conclude that the presence of TSPO in other cell types than microglia, such as astrocytes and endothelial cells is increasingly accepted. We find that the cellular TSPO is not only dependent on the pathology but also on the developmental stage and mode of cell activation. While monitoring TSPO levels in the CNS is now widely used as a readout of microglial activation and neuroinflammation more research is needed to better characterise the means and the cells involved in inflammatory processes and how these processes contribute to the TSPO signal. Future studies could also investigate the therapeutic potential of TSPO manipulation in the CNS.
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