Previous EM observations indicate that Lgi1 knockout impairs myelination in the PNS and CNS (Silva et al., 2010), however, the mechanisms underlying the function of Lgi1 in oligodendrocyte myelination remain elusive. receptor (PDGFR) was from Santa Cruz. Ki67 was from Abcam. Triiodothyronine (T3) and PDGF-AA were from Sigma. Horseradish peroxidase-conjugated secondary antibodies for immunoblotting were from GE Healthcare. IgG antibody, Dulbeccos altered Eagles medium (DMEM), 4,6-diamidino-2-phenylindole (DAPI), Alexa Fluor-conjugated secondary antibodies, neurobasal and B27 supplements were from Invitrogen. FBS was from GIBCO. Other chemicals were from Sigma unless stated otherwise. OPC Culture Purified OPCs from SD rats were isolated by shaking off as explained previously (Zhou et al., 2014). In brief, OPCs were collected from glial cultures by shaking for 1 h at 200 rpm, incubating in new medium for 4 h, and shaking at 250 rpm at 37C for 16 h. Collected OPCs in the medium were re-plated onto poly-D-lysine-coated plates and grew in Neurobasal medium supplemented with 2% B27. PDGF-AA (10 nM) was added in the medium to keep OPCs undifferentiated. Alternatively, T3 (40 ng/ml) was added to the medium for 3 days to allow their differentiation (Li et al., 2013; Zhou et al., 2014). Lentivirus Construction and Transfection Lentivirus encoding small hairpin RNA (shRNA) for Lgi1 (sequence: 5-CCT AAG AGG GAA CTC ATT T-3) was prepared by OBIO (Shanghai). Overexpressing Lgi1 was based on the coding sequence of rat Lgi1 gene (GenBank accession number 145769). Lgi1-shRNA and scrambled RNA were driven by U6 promoter, whereas overexpressed MI-2 (Menin-MLL inhibitor 2) Lgi1 was driven by CMV promoter. OPCs were transfected with 0.05. = 18), tumor infiltration (= DLEU1 18), and tumor mass (= 18), which were confirmed by levels of GFAP and PSD95 in MI-2 (Menin-MLL inhibitor 2) western blot (Physique ?(Figure1D).1D). In consistent with previous statement (Besleaga et al., 2003), our results showed Lgi1 expression was significantly attenuated in gliomas (Figures 1E,F). In the mean time, the expressions of myelin-associated proteins (MBP, MOG, MAG and CNP) were unanimously reduced in gliomas (Figures 1E,F). Hence, these results provide evidence showing the correlated expression between Lgi1 and myelin proteins. Open in a separate window Physique 1 Correlated expression between Leucine-rich glioma inactivated 1 (Lgi1) and MBP. (A) The expressions of Lgi1 and MBP in the brain and spinal cord (SC) from postnatal mice. (B) Lgi1 expression was significantly enhanced in of oligodendrocytes (OLs) compared with in oligodendrocyte precursorcells (OPCs). (C) The immunocytochemical staining of Lgi1 and Olig2 in OPCs and OLs. Level bars, 20 m. (D) An example showing the expression of PSD95 and GFAP in para-cancerous tissue (ctrl), tumor infiltration (TI), and tumor mass (TM) from one patient. The experiment was repeated for 18 occasions as the samples were from six patients. Histogram shows percentage changes of PSD95 and GFAP relative to GAPDH, the loading control. * 0.05, ** 0.01. (E) Western blots of protein expression in isolated tumor infiltration and corresponding control tissues from six patients. Each band represents a sample from one patient. The experiment was repeated for MI-2 (Menin-MLL inhibitor 2) three times. (F) Bar graphs show the percentage changes of MBP, myelin associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), cyclic nucleotide 3-phosphodiesterase (CNP) and Lgi1 after normalized to ctrl. MBP, *** 0.001. MAG, **= 0.0048. MOG, *** 0.001. CNP, *** 0.001. Lgi1, **= 0.0096. Lgi1 Deficiency Causes Hypomyelination in CNS We next used EM to compare myelin formation in the SC from wild-type (WT) and = 4): 0.77 0.02 (WT) and 0.83 0.02 (= 4). (C) EM studies in SC MI-2 (Menin-MLL inhibitor 2) from WT and = 4): 0.77 0.01 (WT) and 0.80 0.01 (= 4). (E) Western blot of myelin protein expression in corpus callosum (CC), optic nerve (ON), cerebellum (CB), and SC from WT and KO mice (P14). CTX: = 4. ON: = 4. CB: = 4. SC: = 4. (F) MBP staining reveals a dramatic reduction of MBP-positive fibers in CC of 0.05, ** 0.01, *** 0.001, ns: no significance. Lgi1 Ablation Impairs OPC Differentiation in the Brain To determine how Lgi1 deficiency causes hypomyelination, we analyzed the expression of cellular markers selected for OPC and OL. Our results showed that the total quantity of OLs indicated by Olig2+ cells in.