Fig 2: p75 is required for MAG-induced dephosphorylation of TrkB and SHP activation. (a) Western blots examining the level of TrkB tyrosine phosphorylation in transfected COS-7 cells. The cells were transfected with the indicated constructs and treated with or without MAG-Fc (25 μg/ml) for 30 min in the presence of 100 ng/ml BDNF for 5 min. Anti-Trk antibodies were used to immunoprecipitate Trk, and the level of Trk phosphorylation was determined by western blotting with anti-phospho-Tyr antibodies. (b–d) Western blots showing the level of TrkB tyrosine phosphorylation in (b) dissociated retinal neurons from WT and p75 KO mice, (c) transfected COS-7 cells co-expressing TrkB FL-HA and PIR-B FL in combination with p75 FL-Myc or p75 ECD-Myc, and (d) COS-7 cells transfected with or without a construct expressing catalytically inactive SHP-2 (SHP-2 C459S). (e) Western blots showing the expression of the indicated proteins in WT and p75 KO CGNs. (f) Western blots for detecting the levels of phospho-SHP-2 (pTyr-580) and total SHP-2 in CGNs following treatment with MAG-Fc for the indicated periods. FGF (50 ng/ml) was used as a positive control. The relative intensity for the phosphorylated SHP-2 signal is indicated (mean: n=3). (g) Western blots showing phospho-SHP-2 (pTyr-580) and total SHP-2 levels in WT and p75 CGNs. The graph shows relative phospho-SHP-2 levels. (h) Relative SHP-2 PTP activity as measured by PTP assays in CGNs from WT (left) and p75 KO (right) mice. Cells were treated with MAG-Fc for 20 min. *P<0.05; Welch's t-test

Fig 3: Interactions between PIR-B, TrkB, and p75 in CGNs. (a) CGNs immunostained for p75 (green), PIR-B (red), and TrkB (blue). Scale Bar: 10 μm. (b and c) Co-immunoprecipitation of the Trk receptors, p75, and PIR-B in COS-7 cells co-expressing combinations of PIR-B FL, and/or p75 FL-Myc with (b) TrkB FL-HA or (c) TrkA FL-HA. Lysates were immunoprecipitated with the indicated antibodies, followed by western blotting. IP, immunoprecipitation; WB, western blot. (d) Co-immunoprecipitation assay of CGNs treated with or without MAG-Fc or OMgp for 15 min. Lysates were immunoprecipitated with the indicated antibodies, and western blotting was carried out. (e) Co-immunoprecipitation of TrkB and PIR-B from CGNs obtained from WT and p75-deficient mice (p75 KO). CGNs were treated with or without MAG-Fc for 15 min. (f) Co-immunoprecipitation of exogenously expressed TrkB-HA and p75 FL-Myc with PIR-B FL from COS-7 cells. The TrkB-HA expression vectors used were WT TrkB, or TrkB with mutations at Tyr-515 (Tyr-515F), Tyr-705/706 (Tyr-705/706F), or both (Tyr-515/705/706F). Lysates were immunoprecipitated with anti-PIR-A/B antibodies, followed by western blotting with the indicated antibodies

Fig 4: Cognitive function and the expression of synaptic proteins and PirB in the hippocampus of CSR mice. a The experimental protocol for CSR is presented. b left, c Representative Y maze test trajectories of control and CSR mice. b right, d Representative NOR test trajectories of control and CSR mice. e, f The percent of spontaneous alteration rates and total number of arm entries in the Y maze test were measured (n = 10). g, h The recognition index and total travel distance in the NOR test were measured (n = 10). i Western blot analysis of hippocampal PSD95 and SYP levels in control and CSR mice. j, k Ratio of fold change from Western blot. The PSD95 (n = 5) and SYP (n = 5) levels were significantly lower in CSR mice than in control mice. l–o Superresolution SIM images and quantification of PSD95 (red, n = 19–20) and SYP (green, n = 19–20) immunoreactive puncta and their apposition (n = 19–20) using Imaris in the hippocampal CA1 region of the two groups of mice. Scale bars: 5 μm. p Western blot analysis of hippocampal PirB levels in control and CSR mice. q Ratio of fold change from Western blot. The PirB level was significantly higher in CSR mice than in control mice (n = 5). r PirB was upregulated in the hippocampus at the mRNA level after CSR (n = 6). s, t Representative immunofluorescence images and quantification of PirB (green, n = 10) fluorescence intensity of the two groups of mice. Scale bars: 20 μm. Asterisks indicate significant differences between groups (2-tailed, unpaired t test, *p < 0.05, **p < 0.01, ****p < 0.0001). Data are presented as the means ± SD

Fig 5: Effect of PirB knockdown on synaptic transmission in the hippocampal CA1 region. a Representative voltage clamp traces show mEPSCs recorded from hippocampal CA1 excitatory pyramidal neurons in slices from four groups of mice at a holding potential of − 70 mV (red: CON, blue: CSR, yellow: PirB cKO; purple: PirB cKO + CSR). b Cumulative probability representing the amplitude of mEPSCs in hippocampal CA1 excitatory pyramidal neurons from four groups of mice. c Cumulative probability represents the frequency of mEPSCs in hippocampal CA1 slices from four groups of mice. d Amplitudes of mEPSCs recorded in slices from the CON mice (red: n = 10 neurons from 5 mice), CSR mice (blue: n = 10 neurons from 5 mice), PirB cKO mice (yellow: n = 10 neurons from 5 mice), and PirB cKO + CSR mice (purple: n = 10 neurons from 5 mice). e Frequency of mEPSCs recorded in slices from the CON mice (red: n = 10 neurons from 5 mice), CSR mice (blue: n = 10 neurons from 5 mice), PirB cKO mice (yellow: n = 10 neurons from 5 mice), and PirB cKO + CSR mice (purple: n = 10 neurons from 5 mice). Data are presented as the means ± SD and were analysed by one-way ANOVA with Tukey’s test (*p < 0.05, ***p < 0.001, ****p < 0.0001)