Fig 2: Recombinant hB7-1–Fc induces synapse remodeling similarly to proNGF.(A) Representative IF images of WT hippocampal neurons treated for 2 hours with proNGF (10 nM), hB7-1–Fc (750 nM), or hB7-2–Fc (750 nM) and stained for actin, PSD95, and MAP2. (B) Quantification of PSD95 puncta density with indicated treatments. Recombinant hB7-1–Fc but not hB7-2–Fc induced a decrease in PSD95 density in cultured neurons, similarly to proNGF treatment. (C) Representative IF images of p75–/– hippocampal neurons treated with proNGF (10 nM), hB7-1–Fc (750 nM), or hB7-2–Fc (750 nM) and stained for actin, PSD95, and MAP2. (D) Quantification of PSD95 puncta density with indicated treatment. Recombinant hB7-1–Fc failed to induce a decrease in PSD95 density in cultured neurons. (E) Representative IF images of WT hippocampal neurons treated with hB7-1–Fc (750 nM) or hB7-2–Fc (750 nM) and stained for actin, GluR1, bassoon, and MAP2. (F) Quantification of bassoon puncta density with indicated treatment. (G) Quantification of GluR1 puncta density with indicated treatment. **P < 0.01, 1-way ANOVA with Dunnett’s multiple-comparisons test. n = 3 independent experiments. Experiments under each condition were performed in triplicate, and 13 to 15 dendritic segments were analyzed per condition. Scale bar: 20 µm. Error bars represent SEM. See also Supplemental Figure 7.

Fig 3: Intranasal Infusion of p75NTR Pen-siRNA Reduces Damage Following CCI. (A) Infusion of p75NTR Pen-siRNA reduced p75NTR expression in the OB and cortex as measured by Western blot. Controls received luciferase Pen-siRNA. Values represent the means ± SEM. Asterisks indicate significance by two-tailed, unpaired Student’s t test with p = .04 for OB and p = .01 for cortex. n = 3 mice/treatment. (B) Adult mice were subjected to CCI and immediately infused intranasally with either p75NTR Pen-siRNA or luciferase Pen-siRNA control. Representative images of cresyl violet-stained coronal sections from luciferase or p75NTR Pen-siRNA-treated mice marked with their coordinates to bregma. Scale bar = 1 mm. The percentage of the area of damage (region of tissue loss and penumbra, indicated by the dotted line) shows less damage in the mice that received the p75NTR Pen-siRNA. Values represent the means ± SEM. Asterisks indicate significance by two-tailed, unpaired Student’s t test with p = .03. n = 3 mice/treatment.p75NTR = p75 neurotrophin receptor; OB = olfactory bulb.

Fig 4: SCID mouse heterotopic xenograft model. A. pLKO cells were subcutaneously injected into SCID mice (n=15 mice per group)Tumors were developed at week 1. One week after pLKO cell xenografts, animals were treated by local injection with PBS/hydrogel (control) or 30μg of pLKO or sh YKL-40 exosomes/hydrogel twice per week during three weeks. Tumor volume and tumor weights were determined at the end of the treatment. Data are presented as the mean ± SD, **P<.01 compared to PBS controls by ANOVA. B. Proliferation visualized by Ki67 staining decreased in pLKO-derived tumor treated by sh YKL-40 exosomes. Immunohistochemical stainings performed in xenograft tumors showed a similar decrease in TrkB, Sortilin, p75NTR, Oct-4 and endothelial marker CD31 expression. Scale Bar, 50μm.

Fig 5: p75NTR:hB7-1 interactions depend on the GFCC′C′′ face of hB7-1 and directly compete with CD28 and CTLA-4.(A) hB7-1 residues that when mutated showed more than 25% losses in binding to CD28 (blue) or both CD28 and CTLA-4 (green) mapped onto crystal structure of hB7-1 (PDB: 1I8L) (B) hB7-1 residues that when mutated showed more than 25% losses in binding to p75NTR (red) mapped onto crystal structure of hB7-1 (PDB: 1I8L). (C) Same residues as in B, except with monomer of CTLA-4 shown. (D) CD28-hIgG1 competes for binding to B7-1 with p75NTR. n = 6. (E) CTLA-4–mIgG2A competes for binding to B7-1 with p75NTR. n = 3. Each assay represents the indicated number of independent experiments, which each include a single replicate. Error bars represent SEM.