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Mechanisms of myelin uptake

Mechanism of myelin phagocytosis in MS
Debbie Hendrickx, PhD student

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) characterized by massive uptake of myelin by microglia and infiltrating macrophages. Fragment crystallizable receptors (FcR) and complement receptor 3 (CR3) mediate myelin phagocytosis, but are not exclusively responsible. Earlier research suggests that scavenger receptors (SRs) might be involved in myelin phagocytosis as they are known to have multiple ligands including oxidized lipids and altered proteins. Also, myelin from MS patients shows enhanced oxidation compared to myelin from healthy controls (HC), thereby possibly creating new epitopes for SRs.

Our aim is to identify receptors that are involved in demyelination with a special focus on SRs. Furthermore, we compare the uptake of MS and HC myelin by the human macrophage cell line, THP-1 and by primary human microglia isolated from fresh post-mortem brain tissue to see if differences in MS myelin could increase its uptake by phagocytes. Myelin is also isolated from human brain tissue and labeled with a special dye that only becomes fluorescent after phagocytosis (see figure 1).

Figure 1. Myelin phagocytosis by primary human microglia. Red: pHrodo-labeled myelin; green: phalloidin/actin; blue: Hoechst/nucleus.
Figure 1. Myelin phagocytosis by primary human microglia. Red: pHrodo-labeled myelin; green: phalloidin/actin; blue: Hoechst/nucleus.

Using laser dissection microscopy (LDM), qPCR and immunohistochemistry, we found that there is a selective upregulation of SRs in and around demyelinated areas in MS, indicating their involvement in myelin phagocytosis.

Figure 2. Selective upregulation of SRs gene expression in and around chronic active and inactive MS lesions.
Figure 2. Selective upregulation of SRs gene expression in and around chronic active and inactive MS lesions.

We are now able to knockdown target genes in THP-1 cells using locked RNA’s and functional studies to confirm the role of these SRs during myelin phagocytosis in vitro are on the way.
Uptake of MS and HC myelin by THP-1 cells and primary human microglia was evaluated using FACS analysis and preliminary results show that MS myelin is indeed taken up more efficiently by both cell types.