Microglia Phenotyping in MS

Marlijn van der Poel, Msc

Microglia are the resident phagocytic cells of the central nervous system and play an important role in brain homeostasis. We study the role of microglia in multiple sclerosis (MS) lesion initiation, since they might be key players in early demyelinating events. We focus on normal appearing tissue, which already shows subtle molecular changes1,2. Previously, our group showed that microglia in normal appearing white matter (NAWM) tissue are already in an alerted state3, and we propose that microglia need two stimuli to become fully activated as seen in MS lesions (Figure 1). We study the complete process on how microglia become fully activated, by identifying the transcriptional profile of alerted human microglia in normal appearing tissue and the effect of a second stimulus on these alerted microglia.

Figure 1: Two-hit hypothesis of microglia activation in MS

Using a protocol optimized in our group4 we isolated primary microglia from normal appearing grey and white matter tissue of 10 MS donors and 11 control donors and determined their gene expression profile by RNA-sequencing. Principal component analysis showed clustering of 4 groups, with highest variation between microglia from grey and white matter brain regions in both MS and control donors (Figure 2). Enriched pathway analysis revealed increased expression of genes involved in lipid metabolism in alerted microglia from NAWM and iron homeostasis and glycolysis pathways in normal appearing grey matter (NAGM) of MS. Thus, changes associated with MS in normal appearing tissue are region specific. Microglia from NAWM already show signs of lipid processing, indicating early demyelinating events in NAWM. However, microglia in NAGM show no signs of lipid processing, indicating that alerted microglia in grey and white matter regions might play a different role in MS lesion initiation.

Next, we will focus on microglia clusters in normal appearing tissue, by identifying the transcriptional profile of specifically these clusters isolated by laser dissection microscopy, as we propose that these clusters are the starting points of MS lesion initiation. In addition, we aim to study the effect of a second hit that might induce full activation of alerted microglia, as we hypothesize that CD8+ T cells or complexed IgG will fully activate microglia (Figure 1).

Figure 2: Principal component analysis of RNA sequencing data shows clear clustering of 4 groups, profound differences were observed between grey and white matter regions in both MS and control donors.

1. Ramaglia et al., PNAS 2012 109(3):965-70
2. Hendrickx et al., Front Immunol 2017 21;8:1810
3. Melief et al., Glia 2013 61(11):1848-61
4. Mizee et al., Acta Neuropathol Commun 2017 17;5(1):16