Journal: Frontiers in Pharmacology

Article Title: BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis

doi: 10.3389/fphar.2016.00512

Figure Lengend Snippet: BNN27 binds directly to p75 NTR receptor. (A) Competition binding assays of [ 3 H]-DHEA in the presence of increasing concentrations of BNN27, using membranes isolated from HEK293 cells transfected with the cDNAs of full-length p75 NTR or ECD-truncated p75 NTR ΔECD receptors (Ki: mean ± SEM of five independent experiments). Right panel depicts efficacy of transfection, assessed by Western blots. (B) Immobilized BNN27 pulls down p75 NTR receptor. Covalently linked BNN27-7- O -(carboxymethyl) oxime (BNN27-7-CMO) to polyethylene glycol amino resin (NovaPEG amino resin) was incubated with recombinant p75 NTR proteins, then centrifuged. Precipitation experiments show Western blot analysis of pellets and supernatants with specific antibodies against p75 NTR proteins, as described in Section “Materials and Methods.” (C, i) STD-NMR revealed the interaction between BNN27 and p75 NTR , further amplified upon NGF additions. No interaction of BNN27 with NGF alone was observed as suggested by the null STD amplification. (ii,iii) 1 H STD-NMR and the corresponding STD-reference spectra. The binding of BNN27 at p75 NTR is evidenced by the presence of BNN27 methyls (18, 19) at the STD spectra; their absence in the case of NGF indicates the lack of BNN27-NGF interaction.

Article Snippet: The recombinant mouse NGFR/TNFRSF16-Fc chimera (also named p75 neurotrophin receptor) comprising the mouse NGFR ECD were purchased by R&D Systems, Inc. NGF (NGF 2.5S, mouse) was purchased by Millipore.

Techniques: Binding Assay, Isolation, Transfection, Western Blot, Incubation, Recombinant, Amplification

Journal: Frontiers in Pharmacology

Article Title: BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis

doi: 10.3389/fphar.2016.00512

Figure Lengend Snippet: Superimposition of representative MD simulations snapshots of BNN27 bound at p75 NTR /NGF 2:1 and at p75 NTR /mutated proNGF 2:2 complexes. Superimposed frames of MD simulations performed at the p75 NTR /NGF 1:2 and p75 NTR /mutated proNGF 2:2 complexes with BNN27. The steroid analog (in yellow) is spontaneously inserted at the CRD4 interfacial region of p75 NTR (in blue)/NGF (in pink). At the modified p75 NTR (in brown)/mutated proNGF (in green), BNN27 (in red) spontaneously penetrates at the interfacial region located at p75 NTR CRD1-CRD2 junction.

Article Snippet: The recombinant mouse NGFR/TNFRSF16-Fc chimera (also named p75 neurotrophin receptor) comprising the mouse NGFR ECD were purchased by R&D Systems, Inc. NGF (NGF 2.5S, mouse) was purchased by Millipore.

Techniques: Modification

Journal: Frontiers in Pharmacology

Article Title: BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis

doi: 10.3389/fphar.2016.00512

Figure Lengend Snippet: BNN27 activates p75 NTR signaling. (A) BNN27 induced the release of RhoGDI from p75 NTR in mouse embryonic fibroblasts (MEF cells). MEF cells were transfected with the plasmid cDNA of p75 NTR 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 p75 NTR -specific antibodies, then immunoblotted with antibodies against RhoGDI. Total lysates were analyzed for p75 NTR 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 p75 NTR in a dose-dependent manner. MEF cells were transfected with the plasmid cDNA of p75 NTR 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 p75 NTR -specific antibodies, then immunoblotted with antibodies against RhoGDI. Total lysates were analyzed for p75 NTR 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 p75 NTR with its effectors RIP2 in MEF cells. MEF cells were transfected with the plasmid cDNA of p75 NTR . 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 p75 NTR -specific antibodies, and then immunoblotted with antibodies against RIP2. Total lysates were analyzed for p75 NTR 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 p75 NTR. MEF cells were transfected with the plasmid cDNAs of p75 NTR wt or p75 NTR 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 p75 NTR -specific antibodies, and then immunoblotted with antibodies against RIP2. Total lysates were analyzed for p75 NTR 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.

Article Snippet: The recombinant mouse NGFR/TNFRSF16-Fc chimera (also named p75 neurotrophin receptor) comprising the mouse NGFR ECD were purchased by R&D Systems, Inc. NGF (NGF 2.5S, mouse) was purchased by Millipore.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Western Blot, Activity Assay, Positive Control

Journal: Frontiers in Pharmacology

Article Title: BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis

doi: 10.3389/fphar.2016.00512

Figure Lengend Snippet: BNN27 protects against apoptosis primary mouse cerebellar granule neurons. (A) Neurotrophin receptors expression in cerebellar granule neuron cultures. Cerebellar granule neurons at 1 DIV express the pan-neurotropic receptor p75 NTR and TrkB, the selective receptor for BDNF, but they do not express TrkA. HEK293 cells transfected with the appropriate cDNA of neurotrophin receptors were used as positive controls. (B) Cell death in response to BNN27 treatment was assessed in p75 NTR wt (up-right) and p75 NTR knockout (KO) (down-right) cerebellar granule neurons (CGNs), identified by b-III tubulin immunostaining (green), using the TUNEL method (red). White arrows indicate TUNEL positive cells that do not express β-III tubulin (red nucleus) and thus represent a non- specific cell population whereas yellow arrows represent TUNEL and β-III tubulin double-positive cells (yellowish nucleus), which is the cell population that was count. Scale bar: 50 μm. (C) Quantification of TUNEL positive neurons CGN cultures (p75 NTR wt, left and p75 NTR KO, right) after 16 h serum withdrawal, incubation with BNN27, NGF or BDNF. Results are expressed as the mean ± SEM. (corrected to control) of three independent experiments, each performed in triplicate. ∗ P < 0.05 versus control (Student’s t -test).

Article Snippet: The recombinant mouse NGFR/TNFRSF16-Fc chimera (also named p75 neurotrophin receptor) comprising the mouse NGFR ECD were purchased by R&D Systems, Inc. NGF (NGF 2.5S, mouse) was purchased by Millipore.

Techniques: Expressing, Transfection, Knock-Out, Immunostaining, TUNEL Assay, Incubation

Journal: Frontiers in Pharmacology

Article Title: BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis

doi: 10.3389/fphar.2016.00512

Figure Lengend Snippet: BNN27 decreases cell death signaling in primary mouse cerebellar granule neurons. (A) Co-immunoprecipitation of RIP2 with p75 NTR in CGNs isolated from p75 NTR wt mice. Incubation of CGNs for 20 min with BNN27, DHEA and NGF induce the recruitment of RIP2 from p75 NTR . Fold change was calculated by densitometric scanning of RIP2 (beads) signals normalized to RIP2 (lysates) levels. Similar results were obtained in three independent experiments. (B) Co-immunoprecipitation of RhoGDI with p75 NTR in CGNs isolated from p75 NTR wt mice. Incubation of CGNs for 30 min with BNN27, DHEA and NGF induce the release of RhoGDI from p75 NTR . Fold change was calculated by densitometric scanning of RhoGDI (beads) signals normalized to RhoGDI (lysates) levels. Similar results were obtained in three independent experiments. (C) Levels of phosphorylated c-jun kinase (p-JNK) in CGNs isolated from wt and p75 NTR KO mice. BNN27, DHEA or NGF Fold change was calculated by densitometric scanning of phospho-JNK signals normalized to total JNK levels. Results are representative of three experiments. (D) Activated Caspase-3 in CGNs, isolated from wild-type (WT) and p75 NTR knockout (KO) mice. After 16 h serum withdrawal treatment. Fold change was calculated by densitometric scanning of cleaved-Caspase-3 signals normalized to actin levels.

Article Snippet: The recombinant mouse NGFR/TNFRSF16-Fc chimera (also named p75 neurotrophin receptor) comprising the mouse NGFR ECD were purchased by R&D Systems, Inc. NGF (NGF 2.5S, mouse) was purchased by Millipore.

Techniques: Immunoprecipitation, Isolation, Incubation, Knock-Out

Journal: Frontiers in Pharmacology

Article Title: BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis

doi: 10.3389/fphar.2016.00512

Figure Lengend Snippet: Schematic illustration of the pro-survival effects of BNN27 in a p75 NTR -mediated manner in primary mouse cerebellar granule neurons. BNN27 activates p75 NTR receptors, leading to the control of pro-survival RhoGDI and RIP2 effectors while in parallel attenuating the activation of pro-apoptotic JNK and Caspase-3 factors.

Article Snippet: The recombinant mouse NGFR/TNFRSF16-Fc chimera (also named p75 neurotrophin receptor) comprising the mouse NGFR ECD were purchased by R&D Systems, Inc. NGF (NGF 2.5S, mouse) was purchased by Millipore.

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Soluble β-amyloid Precursor Protein Alpha Binds to p75 Neurotrophin Receptor to Promote Neurite Outgrowth

doi: 10.1371/journal.pone.0082321

Figure Lengend Snippet: (A) Schematic representations of APP fragments. aa: amino acids. (B–D) Pull-down assays to assess the interaction of APP fragments with p75 NTR . His-tagged sAPPα (B), sAPPβ (C), and C-sAPPα (D) protein were precipitated with Ni-agarose beads. p75 NTR ECD-Fc was co-precipitated with APP fragments. (E–G) Binding of recombinant APP fragments to p75 NTR on p75 NTR -pcDNA transfected COS-7 cells. The cells were transfected with p75 NTR inserted plasmid or control plasmid, and the binding of sAPPα (E), sAPPβ (F), or C-sAPPα (G) on the cells was assessed by immunocytochemistry. Scale bar: 100 µm.

Article Snippet: His-tagged sAPPα, sAPPβ, or C-sAPPα, and Ni-agarose were incubated in binding buffer (HBSS with 0.2% BSA, 0.1% NaN 3 , 5 mM CaCl 2 , 1 mM MgCl 2 , 20 mM HEPES, pH 7.0) at 4°C for 1 h. Human p75 NTR ECD-Fc (1157-NR, R&D Systems) or human IgG-Fc (110-HG, R&D Systems) was added to the solution, and it was incubated at 4°C overnight.

Techniques: Binding Assay, Recombinant, Transfection, Plasmid Preparation, Control, Immunocytochemistry

Journal: PLoS ONE

Article Title: Soluble β-amyloid Precursor Protein Alpha Binds to p75 Neurotrophin Receptor to Promote Neurite Outgrowth

doi: 10.1371/journal.pone.0082321

Figure Lengend Snippet: (A, C, E) ELISA for the p75 NTR -APP fragments interaction. sAPPα (A), sAPPβ (C), or C-sAPPα (E) was plated. After washing with PBS, p75 NTR ECD-Fc or IgG-Fc as a control was added to the plate at the indicated concentrations. The mean OD value after adding p75 NTR ECD-Fc to ELISA microwells coated with recombinant each APP peptides was higher than that of the controls. n = 3. (B, D, F) The sigmoid dose-response curve revealed the EC 50 for each APP fragment-p75 NTR interaction. The EC 50 of sAPPα (B), sAPPβ (D), and C-sAPPα (F) to p75 NTR were 90, 120, 150 nM, respectively.

Article Snippet: His-tagged sAPPα, sAPPβ, or C-sAPPα, and Ni-agarose were incubated in binding buffer (HBSS with 0.2% BSA, 0.1% NaN 3 , 5 mM CaCl 2 , 1 mM MgCl 2 , 20 mM HEPES, pH 7.0) at 4°C for 1 h. Human p75 NTR ECD-Fc (1157-NR, R&D Systems) or human IgG-Fc (110-HG, R&D Systems) was added to the solution, and it was incubated at 4°C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Recombinant

Journal: PLoS ONE

Article Title: Soluble β-amyloid Precursor Protein Alpha Binds to p75 Neurotrophin Receptor to Promote Neurite Outgrowth

doi: 10.1371/journal.pone.0082321

Figure Lengend Snippet: (A) p75 NTR siRNA specifically reduced target protein expression. Cortical neurons were transfected with scrambled control or p75 NTR siRNA. Cell lysates were prepared 72 h after transfection and subjected to western blotting. β-actin expression was used as an internal control. (B, C) siRNA-mediated knockdown of endogenous p75 NTR suppressed sAPPα-induced neurite outgrowth. (B) Representative images of cortical neurons are displayed. Cortical neurons were transfected with scramble siRNA (control) or p75 NTR siRNA. Three days after transfection, the neurons were incubated in the presence or absence of sAPPα for 24 h. Scale bar: 100 µm. (C) The mean lengths of the longest neurite per neuron were measured by image J software and represented in the graph. The graph showed the mean ± SEM from of three independent experiments. The number of neurons was 150 for each experiment. ** p <0.01, Scheffe's F test.

Article Snippet: His-tagged sAPPα, sAPPβ, or C-sAPPα, and Ni-agarose were incubated in binding buffer (HBSS with 0.2% BSA, 0.1% NaN 3 , 5 mM CaCl 2 , 1 mM MgCl 2 , 20 mM HEPES, pH 7.0) at 4°C for 1 h. Human p75 NTR ECD-Fc (1157-NR, R&D Systems) or human IgG-Fc (110-HG, R&D Systems) was added to the solution, and it was incubated at 4°C overnight.

Techniques: Expressing, Transfection, Control, Western Blot, Knockdown, Incubation, Software