GAIN domain and ADGRG1N-hFc are shown. for OL lineage cells is unknown. Here, we report that microglia-derived transglutaminase-2 (TG2) signals to ADGRG1 on OL precursor cells (OPCs) in the presence of the ECM protein laminin and that TG2/laminin-dependent activation of ADGRG1 promotes OPC proliferation. Signaling by TG2/laminin to ADGRG1 on OPCs additionally improves remyelination in two murine models of Efnb2 demyelination. These findings identify a novel glia-to-glia signaling pathway that promotes myelin formation and repair, and suggest new strategies to enhance remyelination. cause the devastating human brain malformation called?bilateral frontoparietal polymicrogyria (BFPP), which is comprised of a constellation of structural brain defects including CNS hypomyelination (Piao et al., 2004; Piao et al., 2005). Conditional deletion of in OL lineage cells results in CNS hypomyelination, and this is specifically caused by deficiencies in ADGRG1 signaling in OPCs and immature OLs (Giera et al., 2015). Loss of in mice and zebrafish decreases OPC proliferation, thereby leading to a reduced number of mature myelinating OLs and fewer myelinated axons in the CNS (Ackerman et al., 2015; Giera et al., 2015). However, the relevant ADGRG1 ligand during CNS myelination has not yet been defined. In this study, we demonstrate that microglia-derived transglutaminase 2 (encoded by leads to reduced OPC cell division, fewer mature OLs, and hypomyelination during postnatal CNS development, phenocopying the loss of we find that the activation of ADGRG1 by TG2 requires the ECM protein laminin to promote OPC proliferation. Moreover, we extend our analysis GW1929 of the role ADGRG1-TG2 interactions in developmental myelination to mouse models of myelin damage. We provide evidence that OPC-specific deletion of impairs CNS remyelination after toxin-induced demyelination, and that recombinant TG2 rescues remyelination failure GW1929 in organotypic cerebellar slices in a ADGRG1-dependent manner. Taken together, these findings show that the tripartite signaling complex comprised of microglial TG2, extracellular laminin, and OPC ADGRG1 regulates OL development and myelin repair. Results Putative ligands of ADGRG1 are expressed in microglia aGPCR ligand binding is solely mediated by its N-terminal fragment (NTF)(Hamann et al., 2015). To establish the distribution of an OPC-specific ADGRG1 ligand in the developing brain, we labeled putative ADGRG1 binding proteins in mouse corpus callosum (CC) tissue with a probe comprised of the ADGRG1 NTF fused to human immunoglobulin Fc fragment (ADGRG1N-hFc; Figure 1A). These studies were performed at postnatal day 5 (P5) when OPCs are actively proliferating. To GW1929 identify the lineage of ADGRG1 ligand-expressing cells, we performed a series of double IHC experiments with ADGRG1N-hFc paired with Iba1 (to label microglia), GFAP (to label astrocytes), and PDGFR (to label OPCs). We observed robust and consistent putative ligand detection in microglia (Figure 1B and C), while no obvious putative ligand binding was detected in OPCs and only sparse signals were observed in astrocytes (Figure 1DCG). Quantitative analyses showed that?~80% Iba1?+microglia and~20% of GFAP?+astrocytes express the putative ligand of ADGRG1 (Figure 1H). Open in a separate window Figure 1. Microglia express the putative ligand of ADGRG1.(A) Schema of ADGRG1 receptor. GAIN domain and ADGRG1N-hFc are shown. (B, D, and F) Double labeling of ADGRG1N-hFc (green) and Iba1 (B, red), GFAP (D, red), PDGFR (F, red) in P5 wt corpus callosum. DAPI, blue. Scale bar, 25 m. (C, E, and G) Higher magnification of the boxed region in (B, D and?F). Scale bar, 10 m. Staining was repeated knockout mice display reduced myelination We first focused our attention on TG2 because it is a known binding partner of ADGRG1 in melanoma cells (Xu et al., 2006). To evaluate the candidacy of TG2 as the ligand of ADGRG1 in OPCs, we investigated whether deleting phenocopies the myelination phenotype observed in knockout mice. Indeed, we observed significantly reduced numbers of knockout GW1929 mice at both P14 and P28 compared to littermate controls (Figure 2A and B),.