Fig 1: Characterization and functional validation of brain exosomes from EXE-5XFAD mice. (a) Representative electron micrograph of isolated brain exosomes. Scale bar: 200 nm. (b) Representative results of nanoparticle tracking analysis demonstrating size distribution of brain exosomes. (c) Representative blots of brain exosomes marker proteins CD63 and CD81 and endoplasmic reticulum protein Calnexin showing vesicles purified using differential centrifugation were exosomes. (d) Representative blots of brain exosomes marker proteins CD63 and endoplasmic reticulum protein Calnexin showing exosomes purified using sucrose density gradient centrifugation existed mainly in c–e fractions. (e) Primary pericytes were cultured in the presence or absence of PKH26-labeled exosomes (red) at 37°C for 6 h. Scan bar: 20 µm. (f) Immunoblotting of PDGFRß, NG2, and ZO-1 in cultured primary pericytes and endothelial cells, respectively, treated with PBS, SEX-exo, and EXE-exo. (g) Quantification of PDGFRß, NG2, and ZO-1 relative expression in primary pericytes and endothelial cells treated with PBS, SEX-exo, and EXE-exo respectively. ß-actin was used as a control. N = 3 replicates; Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA. (h and i) MTT assay showed that exosomes isolated from the brain of EXE-5XFAD mice improved cell proliferation both in endothelial cells (h) and pericytes (i). N = 3 replicates; Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA. (j) Primary pericytes and endothelial cells were treated with PBS, SED-exo and EXE-exo for 48 h. Representative micrograph of cell apoptosis determined by PI staining (red). Scan bar: 100 µm. (k, l) Quantification of the apoptosis of primary endothelial cells (k) and pericytes (l). Cell apoptosis was determined as a percentage (%) of PI-positive cells covering all DAPI-positive cells. N = 3 replicates; Mean ± SEM; **p < 0.01, ***p < 0.001 by one-way ANOVA.
Fig 2: (A) Fluorescence micrographs show circumferential distribution of LepR+ cells (red) around HEVs and lymphatics in Leprcre;tdTomato mice LNs. LepR (red) colocalized with PDPN and ER-TR7. Scale bar, 50µm. (B) Fluorescence micrographs of LNs from Leprcre;tdTomato mice showed no colocalization of LepR (red) with NG2 and PDGFRß. Scale bar, 100µm. (C) Fluorescence micrographs of Leprcre;tdTomato mice LNs showed no colocalization of LepR (red) with RANKL, CD35 and MAdCAM. Scale bar, 50µm. (D) LepR expression in CD45-PDPN+CD31-(FRCs) and CD45-PDPN-CD31-(DN) populations of LNs were evaluated by flow cytometry. Data are representative of three independent experiments (n=3). (E) Gating strategy to exclude hematopoietic and endothelial cells for LepR+ cells in LepRcre;tdTomato LNs. (F) The percentages of LepR+ cells of LNs in stromal marker panel Sca-1, CD29, CD90, CD44, CD73, CD105 and CD106 were evaluated based on the gating strategy in (E). Data are representative of three independent experiments (n=3). (G) Fluorescence micrographs of PND1 and PND14 LNs showed location of LepR+ cells (red) in relation to HEVs, lymphatics, PDPN and ER-TR7. Scale bar, 100µm.
Fig 3: Expression of CK2 and NG2 in the patient-derived JA cells. (A) Bright field images (scale bars: 20 µm) and immunofluorescence stainings (scale bars: 50 µm) of NG2 (red), vimentin (green) and cell nuclei (blue) in the JA1–JA4 cells. (B) The JA1–JA4 cells were lysed and the expression of NG2, CK2a, CK2ß and a-tubulin (as a loading control) was analyzed by Western blot. (C–E) Quantitative analysis of (B). Data are expressed as the relative density ratio of NG2/a-tubulin (D), CK2a/a-tubulin (E) and CK2ß/a-tubulin. (F) NG2 surface expression of the JA1–JA4 cells was detected by flow cytometry. Mean ± SD (n = 2). (G) RNA was extracted from the JA1–JA4 cells and the gene expression of NG2 was examined by qRT-PCR (% of JA1). Mean ± SD (n = 3).
Fig 4: Survival and maturation of OLs are modified by A2BAR activation in SCZ mice. (A) Immunohistochemical images of NG2+ OPC staining in the cerebral cortex (scale bar, 50 µm) The arrows and insert box indicate the NG2+ cells; the inset boxes are at x8 magnification. (B) MK-801 treatment markedly reduced the number of NG2+ OPCs in the cerebral cortex. Administration of BAY 60-6583 in the SCZ mice notably increased the number of NG2+ OPCs in the cerebral cortex. (C) Representative immunoblots of the NG2 protein levels. (D) MK-801-treated mice showed a significant reduction in NG2, while BAY 60-6583 administration increased NG2 protein levels in the cerebral cortex. (E) Representative immunoblots of GPR17 protein expression levels. (F) GPR17 protein expression levels were significantly increased in MK-801-treated mice and were restored in MK-801 + BAY-treated mice. (G) The number of CC-1+/Olig2+ cells was reduced in the MK-801 group, while a significant increase was observed in the MK-801 + BAY group. (H) Immunofluorescence staining was performed for CC-1/Olig2 in the cerebral cortex (scale bar, 50 µm). Data are presented as the mean ± SEM. *P<0.05. OLs, oligodendrocytes; A2BAR, A2B adenosine receptor; SCZ, schizophrenia; NG2, chondroitin sulfate glycoprotein 4; OPCs, OL precursor cells; MK-801, dizocilpine maleate; GPR17, G protein-coupled receptor 17; CC-1, anti-adenomatous polyposis coli clone CC-1; Olig2, OL transcription factor 2; BAY 60-6583, A2BAR agonist; PSB 603; A2BAR antagonist.
Fig 5: Striatal protein (normalized to the control group level) for CTL, IUGR, and IUGR_Lf pups at P7 (high panel) and P21 (low panel). Structural protein expression at P7 (DCX, NeuN, Synapto., NG2, GFAP, CD68, and Iba1). Expression of striatal pro- and anti-apoptotic mRNA at P7 (Bax and BCL2). Selection of metabolic transporter and receptor expressions in the striatum at P21 (MCT2, NMDar2a, DMT1, CaMKIIß, and Leptin R). Growth factor molecule and receptor mRNA expression in the striatum at P21 (TrKB and IGF2). Results are mean values ± SEM of N = 3 to 14 pups per group. P < 0.05 *CTL vs. IUGR, £IUGR vs. IUGR_Lf. Raw data in Supplementary Tables 4, 5.
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