Transforming growth factor-beta renders ageing microglia inhibitory to oligodendrocyte generation by CNS progenitors

Abstract

It is now well-established that the macrophage and microglial response to CNS demyelination influences remyelination by removing myelin debris and secreting a variety of signaling molecules that influence the behaviour of oligodendrocyte progenitor cells (OPCs). Previous studies have shown that changes in microglia contribute to the age-related decline in the efficiency of remyelination. In this study, we show that microglia increase their expression of the proteoglycan NG2 with age, and that this is associated with an altered micro-niche generated by aged, but not young, microglia that can divert the differentiation OPCs from oligodendrocytes into astrocytes in vitro. We further show that these changes in ageing microglia are generated by exposure to high levels of TGFβ. Thus, our findings suggest that the rising levels of circulating TGFβ known to occur with ageing contribute to the age-related decline in remyelination by impairing the ability of microglia to promote oligodendrocyte differentiation from OPCs, and therefore could be a potential therapeutic target to promote remyelination.

Keywords: ageing; extracellular matrix; microglia; oligodendrocyte; progenitor cells.

Figure 1

TGF β activated microglia surface molecules alter OPC differentiation in vitro. (a) A schematic description of the protocol used for generating microglia ghosts. A2B5 MACS was used to remove OPCs from cell suspension. After overnight recovery, microglia were isolated using CD11B labeled magnetic microbeads. Cells were plated, and lysed using ddH₂O after 48 hr in culture. (b) 80% of the cells cultured from both young and aged adults following CD11B microbeads isolation were either CD11B+ or IBA1+ microglia. (c) Neonatal OPCs (isolated using A2B5 MACS) cultured on young microglia ghosts do not show any change in differentiation capacity (n = 3, p value > .05). (d) Neonatal OPCs cultured on top of neonatal microglia ghosts, show a not significant change in GFAP expression (ratio paired t test, n = 5, p value > .05), with specific culture presenting high percentages of astrocyte formation. (e) Neonatal OPCs cultured on top of TGFβ1 treated young microglia ghosts show significant increased expression of GFAP (one way ANPVA, n = 4, p value < .05) in a dose dependent manner. (f) Representative images of cultured microglia (young and aged). (g) Representative images of OPCs cultured on top of control PDL or aged microglia ghosts. OPCs were cultured for 4–5 days in serum free medium, and were stained for GFAP (red) to identify astrocytes formation and NG2 (white) and O4 (green) to identify OL lineage cells (scale bar represents 100 μm) [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

TGFβ alters the expression of surface molecules in young microglia. (a) Young microglia treated in vitro (48 hr) with TGFβ1 exhibit increase in mRNA expression of multiple surface antigens, such as Fn1, Cspg4, and Cd44, quantified by qRT‐PCR (n = 3). The increase in mRNA expression is eliminated in the presence of the TGFβ small molecule inhibitor (SB‐432541, 5 μM). (b) in vitro quantification of young microglia cultured in the presence of TGFβ1 (20 ng/mL) with or without TGFβ inhibitor (SB‐432541, 5 μM) (n = 4, p value < .01). (c) IHC of microglia treated with TGFβ co‐expressing NG2 (green) and microglia marker IBA1 (red) [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Changes in aged microglia surface molecules. (a) Published RNA‐seq data show increases in expression of multiple proteoglycan genes in aged microglia when compared with young microglia cells (Hickman et al., 2013). (b) FACS isolation of young (2–4 months old) and aged (18–22 months old) microglia using CD11B antibodies reveals a significant increase in microglia proportions in aged rats (paired t test, p value < .05, n = 3). (c) FACS analysis of the proportion of NG2+ cells out of total CD11B+ cells show a significant increase in aged rats (paired t test, p value <0.005, n = 3). (d) Example of FACS scatterplot of aged microglia. (e) Representative images of young and aged microglia cultured in vitro and stained for IBA1, CD11B (microglia markers), and NG2. (f) Small numbers of NG2+/CD11B− cells with a flat morphology were also detected. (g) Proportions of NG2+ are higher in aged microglia acutely isolated using CD11B MACS and cultured 24–48 hr in vitro (paired t test, p value < .05, n = 3). (h) There was no significant difference in the percentage of these cells between aged and young microglia preparations (scale bar represents 100 μm) [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Aged microglia surface molecules alter OPC differentiation in vitro. (a) Neonatal OPCs cultured on top of aged microglia ghosts show significant increased expression of GFAP (ratio paired t test, n = 5, p value < .05). (b) Culturing neonatal OPCs on young or aged microglia ghosts in the presence of BMP inhibitor (LDN‐193189) blocks the differentiation into astrocytes and GFAP expression (ratio paired t test, n = 3, p value < .05). (c) OPCs cultured with aged microglia conditioned media (CM) do not show upregulation in astrocyte formation (p value > .05, n = 3). (d,e) Representative images of OPCs cultured on top of control PDL or aged microglia ghosts. OPCs were cultured for 4–5 days in serum free medium, and were stained for GFAP (green) to identify astrocytes formation (scale bar represents 100 μm) [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

NG2 positive microglia present in NAWM and demyelinating lesions in aged rats. (a) NAWM (normal appearing white matter) in aged rats was imaged for CD11B (identification of microglia) and NG2. Several microglia were double labeled. (b) 5 days after the induction of demyelinating lesion, multiple microglia cells expressed high reactivity to NG2 staining. Green arrows represent CD11B + NG2− cells, red arrows point out CD11B − NG2+ cells and yellow arrows point to CD11B + NG2+ cells [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6

TGFβ directly inhibits OPC differentiation in vitro. (a) OPCs were cultured with various concentrations of TGFβ (0–20 ng/mL) for 6 days in vitro. Differentiation was assessed by CNP staining. The presence of TGFβ in culture media resulted in significant reduction in OPC percentage of CNP+/OLIG2+ cells (out of total OLIG2+ cells, normalized to control) (one‐way ANOVA, n = 3, p value < .05). (b) When cells were cultured with TGFβ and the addition of TGFβ small molecule inhibitor (SB‐431542; 5 μM) differentiation inhibition was eliminated (one‐way ANOVA, n = 3, p value > .05). (c) OPCs cultured with 10 ng/mL TGFβ and the presence of BMP pathway inhibitor (LDN‐193189; 5 μM) showed again significant decrease in differentiation capacities (paired t test, n = 2, p value < .05). (d) Schematic description of pathways blocked by SB‐431542 and LDN‐193189. (e) Representative images of OPCs in vitro stained for OLIG2 (red) and CNP (cyan) with or without the addition of TGFβ [Color figure can be viewed at wileyonlinelibrary.com]