Fig 2: HEK293T cells were transiently co-transfected with the plasmid cDNAs of p75NTR and/or TRAF6 and/or RIP2. (a) Cell tox assay performed on transfected HEK293T cells after 48 h and treatment with the tested compound ENT-A044 (500 nM). (b) Quantification of cell tox+ cells (green)/Hoechst+ cells (blue), one-way ANOVA, * p < 0.05, ** p < 0.01, mean ± SEM of triplicate measurements. (c) Western blot analysis performed on transfected HEK293T cells after 30 min of treatment with the tested compound (500 nM) and quantification of pJNK expression (unpaired t-test against control serum free, * p < 0.05, mean ± SEM of triplicate measurements). (d) Transfectants were exposed for 20 min to BDNF (500 ng/mL), and the tested compound and lysates were immunoprecipitated with p75NTR-specific antibody and immunoblotted with antibodies against TRAF6. Total lysates were analyzed for p75NTR or actin expression via immunoblotting (unpaired t-test against control serum free; ns, no significant; mean ± SEM of triplicate measurements). (e) Transfectants were exposed for 20 min to BDNF (500 ng/mL), and the tested compound and lysates were immunoprecipitated with p75NTR-specific antibody and immunoblotted with antibodies against RIP2. Total lysates were analyzed for p75NTR or actin expression via immunoblotting (unpaired t-test against control serum free, *** p < 0.005, mean ± SEM of triplicate measurements).

Fig 3: BNN27 activates p75NTR signaling. (A) BNN27 induced the release of RhoGDI from p75NTR in mouse embryonic fibroblasts (MEF cells). MEF cells were transfected with the plasmid cDNA of p75NTR and control plasmid (Mock). Transfectants were exposed for 30 min with BNN27 (100 nM), DHEA (100 nM), NGF (100 ng/ml), or Estradiol (E2) (100 nM), and lysates were immunoprecipitated with p75NTR-specific antibodies, then immunoblotted with antibodies against RhoGDI. Total lysates were analyzed for p75NTR expression by immunobloting. Fold change was calculated by densitometric scanning of RhoGDI (beads) signals normalized to RhoGDI (lysates) levels. Results are representative of three independent experiments. (B) Regulation of RhoA activity by BNN27 in MEF cells. Constitutively active RhoA protein (provided by the kit manufacturer) was used as positive control. Results are expressed as the mean of triplicate measurements ± SEM normalized to control. ∗P < 0.05 versus control. (C) BNN27 induced the release of RhoGDI from p75NTR in a dose-dependent manner. MEF cells were transfected with the plasmid cDNA of p75NTR and control plasmid (Mock). Transfectants were exposed for 30 min with various concentrations of BNN27 (1, 10, or 100 nM), or NGF (100 ng/ml), and lysates were immunoprecipitated with p75NTR-specific antibodies, then immunoblotted with antibodies against RhoGDI. Total lysates were analyzed for p75NTR expression by immunobloting. Fold change was calculated by densitometric scanning of RhoGDI (beads) signals normalized to RhoGDI (lysates) levels. Results are representative of three independent experiments. (D) BNN27 induced the association of p75NTR with its effectors RIP2 in MEF cells. MEF cells were transfected with the plasmid cDNA of p75NTR. Transfectants were exposed for 30 min to vector (Control), BNN27 (100 nM), DHEA (100 nM), NGF (100 ng/ml) or Estradiol (E2) (100 nM), and lysates were immunoprecipitated with p75NTR-specific antibodies, and then immunoblotted with antibodies against RIP2. Total lysates were analyzed for p75NTR expression by immunobloting. Fold change was calculated by densitometric scanning of RIP2 (beads) signals normalized to RIP2 (lysates) levels. Similar results were obtained in three independent experiments. (E) Structure-function relationships in the interaction between BNN27 p75NTR. MEF cells were transfected with the plasmid cDNAs of p75NTR wt or p75NTR mutants that lack the entire Extracellular Domain (ΔECD) or p75C257A and interactor RIP2. Transfectants were exposed for 30 min to vector (Control), BNN27 (100 nM), DHEA (100 nM), NGF (100 ng/ml) or E2 (100 nM), and lysates were immunoprecipitated with p75NTR-specific antibodies, and then immunoblotted with antibodies against RIP2. Total lysates were analyzed for p75NTR expression by immunobloting. Fold change was calculated by densitometric scanning of RIP2 (beads) signals normalized to RIP2 (lysates) levels. Similar results were obtained in three independent experiments.

Fig 4: p75NTR expression and function on human iPSCs - derived NSCs. A Representative blots (n = 3) determined via Western Blot showing the expression of p75NTR, RIP2 and TRAF6 proteins in lysates of two iPSCs-derived NSCs lines (841, 856). Co-IP showing the interaction of p75NTR with TRAF6 protein. HEK293T cells, mNSCs p7 (mouse NSCs from postnatal day 7), PC12 (pheochromocytoma of the rat adrenal medulla), HEK293T cells transiently transfected with p75NTR plasmid and NIH3T3 cells expressing TrkB receptor were used like control samples. kDa, molecular weight; M, Marker for molecular weights, p75NTR (70–75 KDa), actin (42 KDa), TRAF6 (60KDa), RIP2 (61KDa). B Dead cells (green) upon inhibition of p75NTR and/or treatment with aβ oligomers (50 μm scale bar). C Graph showing the percentage of dead cells (Celltox labeled cells/total number of HOECHST+ cells) n = 3 from different NSCs lines (841, 856) (one way ANOVA **p < 0,05, ***p < 0,005, ns, no significant)