Fig 1.
A Cell-type specific staining of magnetically sorted (MACS) primary OPC (pOPC) at different timepoints of culture. DIV0, 1 and 2: 2h, 24h and 48h of culture respectively (DIV: days in vitro). OPC were identified by co-expression of NG2 and Olig2 (NG2+/Olig2+), Scale bar = 30μm and 15μm for the zoom. The percentage of the identity of each cell-type of the total DAPI+ cells is given in B. Differentiated oligodendrocytes were identified by the expression of PLP, astrocytes by expression of GFAP, microglia by expression of F4/80 and neurons by expression of ß-III-tubulin (TUJ-1). Pericytes were not detected in in the pOPC culture, see S1 Fig for pericyte staining. (A&B: 200 cells were analyzed for each staining/time-point from 2 independent MACS sorts).
Fig 2.
Expression of PTGDS and Nptx2 in primary OPC.
A Expression of cell-type specific markers was analyzed by Western-Blot of total cell lysates of pOPC and the negative (neg.) sort fractions, HEK cells and primary cortical neurons (DIV5 in culture). B-D show the expression of proteins in pOPC over time, related to DIV0. In B a peak of the OPC protein NG2 is shown at DIV1, PLP indicates differentiation into oligodendrocytes starting at DIV2, GFAP shows astrocyte differentiation starting at DIV2. C PTGDS expression peaks together with NG2 at DIV1. D Nptx2 expression increases together with NG2 at DIV1, peaks at DIV2 and returns to basal levels at DIV4. E Expression of PTGDS and Nptx2 mRNA in pOPC at DIV1, as revealed by in situ hybridization, OPC were identified by antibody staining of NG2 protein. (A-D: 3 sorts were analyzed for each time point. Two tailed student’s t-test was applied.)
Fig 3.
NG2 dependent regulation of PTGDS and Nptx2 in vivo.
A&B mRNA levels of target-genes were directly analyzed by qRT-PCR from FACS isolated OPC and other cells (negative (neg.) sort). Single cell suspensions from total brains of postnatal day 9 (P9) NG2-KO and WT mice were used for FACS. A Enrichment of target mRNA in OPC in comparison to other cell-types is shown for WT (black bar) and NG2-KO (grey bar), (ΔΔCT = [ΔCT OPC]–[ΔCT other cells]). Enrichment of PDGFRα mRNA validates OPC enrichment. Nptx2 mRNA was enriched within OPC of both genotypes, while PTGDS mRNA was only enriched in WT OPC. B Genotype-specific mRNA enrichment in OPC (black bar) and other cells (grey bar) is plotted (ΔΔCT = [ΔCT WT cells]–[ΔCT KO cells]). PTGDS was the sole target gene analyzed exhibiting differential expression between WT and KO genotypes. Expression was highly increased in WT-derived OPC and down-regulated in the other cells (neg. sort) from these mice. C Western Blot of soluble protein fractions. Soluble fractions were extracted from P9 mouse brain of WT and NG2-KO mice. Total PTGDS and Nptx2 protein levels show no difference between genotypes. D Protein levels of post nuclear (PN) cell lysates of the OPC cell line Oli-neu were analyzed after treatment with siRNA silencing NG2 expression (siNG2) or control siRNA (siC). Full-length NG2 levels were reduced as well as PTGDS protein levels, fitting to the reduced mRNA levels of PTGDS found in NG2-KO OPC (B). E Expression of PTGDS mRNA by the OPC cell-line Oli-neu as revealed by in situ hybridization. (A&B: for NG2-KO OPC, 4 independent sorts were analyzed, for WT 3 sorts were analyzed, ΔCT = (CT target)–(CT GAPDH), ΔCT and ΔΔCT values are in log2 scale; for C: 4 animals were analyzed for NG2-KO (KO1-4) and BL6/N (WT1-4) mice; for D: 4 independent transfections were analyzed per siRNA, two tailed student´s t-test was performed.)
Fig 4.
The NG2 ICD is located in the nucleus.
A Cartoon of the NG2 full length protein and the major cleavage fragments (ectodomain, CTF, ICD). Ectodomain cleavage (indicated by the α) has been reported by others [28], while intracellular cleavage (indicated by the γ) has been found by our group [21]. Expression constructs NG2_del leading to high NG2 CTF and lower ICD levels by proteolytic processing and the NG2_ICD construct leading to high levels of NG2 ICD are both shown in red. B Cytoplasmic (cyto), crude membrane (CM) and nuclear fractions of HEK cells transfected with empty plasmid (control), NG2_ICD, or NG2_del are shown. NG2_del full-length (FL) protein, the membrane bound NG2 CTF and the NG2 ICD are shown in WB (schematically shown in Fig 2D). The NG2 ICD shows the highest levels when expressed as a recombinant protein (NG2_ICD). This is present in all fractions but highest in the nuclear fraction. NG2_del-derived NG2 ICD (generated by proteolysis) is only detectable in the nuclear fraction and runs at the same size as the recombinant NG2 ICD.
Fig 5.
NG2 fragments exhibit distinct subcellular localization including the nucleus.
Fluorescent staining of OPC expressing NG2_del_Myc or the NG2_ICD_Myc construct (in red) and GFP tagged Histone2B (H2B-GFP, only A&B). A&B Confocal laser scanning microscope (cLSM) pictures of Oli-neu (OPC cell line) show NG2_del_Myc (red) staining at the plasma membrane and intracellular membranes, but nuclei are largely spared. Expression of the NG2 ICD (NG2_ICD_Myc, red) results in an almost homogeneous cytoplasmic staining including an intense staining of the nuclei. A Standard deviation (STD) projection is shown for the total LSM stack. NG2 ICD (Myc staining) shows much stronger overlap with nuclear stainings (H2B-GFP, DAPI) than the one of NG2_del (scale bar = 20μm). B Three dimensional orthogonal views (ImageJ3DViewer) are shown for cell bodies of the two transfection conditions. NG2 ICD reveals a striking intranuclear staining (overlap with H2B-GFP), while NG2_del only overlaps at the outer layer of the H2B stained nucleus. C Transfected pOPC showed a much lower expression of both NG2 constructs. The strongest staining of Myc-tagged constructs is observed close to the nucleus in NG2_del_Myc transfected cells. In NG2_ICD_myc transfected cells, many cells additionally show a staining of nuclear substructures. Images show one plane from the center of a confocal z-stack (pAB = primary antibody, scale bar = 15μm). For staining of pOPC also see S2B Fig. D Cartoon of the intracellular domain (ICD) of NG2. E Selected NG2 ICD sequences with predicted functions. ELM database analyses show 2 NLS sequences indicating nuclear transport and a predicted WW4 binding motif, a binding site for protein complexes which localize to nuclei. (A&B: 20 double-transfected cells from 3 independent transfections per condition were analyzed; 100% of the NG2_del and over 70% of the NG2_ICD transfected cells showed the described effects. cLSM stacks had a z-stack depth of at least 12 μm, with a z-increment of < 0.3 μm per single image.)
Fig 6.
NG2 intracellular fragments influence PTGDS protein levels.
A Expression of NG2_del, NG2_ICD (both red) and Mock (empty Plasmid) constructs in the OPC cell-line Oli-neu, resulted in a reduction of PTGDS protein levels in post nuclear lysates (PN). Overexpression of these constructs leads to a changed ratio of protein levels between the NG2 FL and the small NG2 (intracellular) cleavage fragments the CTF and the ICD (compare to mock, see B). B Cartoon of NG2, NG2 expression constructs (red) and the cleavage sites for α- and γ-secretase leading to the creation of the CTF (12 kD) and ICD (8.5 kD, both blue). (A: 4 independent transfections were analyzed per construct; two tailed student’s t-test was performed.)