Journal: Frontiers in Immunology

Article Title: Platelets Selectively Regulate the Release of BDNF, But Not That of Its Precursor Protein, proBDNF

doi: 10.3389/fimmu.2020.575607

Figure Lengend Snippet: Human platelets contain proBDNF. (A) ProBDNF immunoblotting of human washed platelet lysates (15 µg) from six different healthy volunteers. Recombinant proBDNF (3 ng) and human cortex lysate (3 µg) were used as positive controls. Molecular weight is indicated on the left (kDa) and primary antibody on the right. Experiments representative of n=9 for R-176 and n=10 for mab31751 antibody. IB, immunoblotting. (B) Immunoblotting of proBDNF and BDNF in different fractions of washed human platelets. P-Selectin was used as control protein in the membrane fraction, p65 NF-ĸB was used as control protein in the cytosol, and α-tubulin was used as control protein in the cytoskeleton. The equivalent of the protein content of 3 x 10 7 platelets was loaded for each fraction on the gel. Representative experiment of n=4 different volunteers. (C) ProBDNF treatment with PNGase F in washed human platelet lysates. U87-MG glioblastoma cells were used as a control. rProBDNF, recombinant proBDNF (3 ng); cortex, human cortex lysate (3 µg); platelets, whole human platelet lysate (representative experiment of n=4 different volunteers, 7.5 x 10 8 platelets per well); −PNGF, platelets treated with GlycoBuffer, and incubated at 37°C for 60 min without PNGase F; +PNGF, platelets treated with GlycoBuffer and incubated at 37°C for 60 min with PNGase F; PNGF alone, PNGase F incubated at 37°C for 60 min without platelet lysate. CD42b and sortilin were used as controls of protein deglycosylation in platelets and in U87-MG cells, respectively. n=3 different volunteers for PNGase treatments in human platelets and n=4 independent experiments for U87-MG cells. (D) Representative flow cytometry experiment showing surface and intracellular proBDNF in human washed platelets and in U87-MG and U251-MG glioblastoma cell lines. Mouse IgG 2b was used as isotype control. Percentage of expression are indicated on the figure. n=10 different healthy volunteers for human platelets; n=3 independent experiments for each glioblastoma cell line. (E) Confocal microscopy imaging of proBDNF in human permeabilized washed platelets (top) and in permeabilized U-251 MG cells (bottom). Mouse IgG 2b was used as isotype control. ProBDNF was labelled using Alexa488 fluorochrome (in green). Nuclei were stained with DAPI (in blue). Scale bar = 2 µm and 200 µm for washed platelets and U-251 MG cells images, respectively. (F) Immunoblotting of α 2 -macroglobulin at increasing quantities of loaded proteins (1–20 µg) obtained from a washed platelet lysate or platelet-poor plasma (PPP) from the same individual (#23). α-tubulin was used as loading control. Molecular weight is indicated on the left (kDa) and primary antibody on the right. PPP, platelet poor plasma; PLTs, platelets; IB, immunoblotting.

Article Snippet: Platelets and neuroblastic cells were then incubated at RT with mouse anti-human proBDNF antibody (R&D System, mab31751, monoclonal mouse antibody, clone 584412, diluted 1:25 or Biosensis, R-176 polyclonal rabbit antibody diluted 1:25) or mouse IgG 2b /rabbit isotype control (R&D System, MAB004 and AB-105-C diluted 1:25) for 30 min. Alexa Fluor 488 or 647 conjugated donkey anti-mouse or rabbit secondary antibody (Invitrogen, diluted 1:100) was added for 30 min at RT in the dark.

Techniques: Western Blot, Recombinant, Molecular Weight, Control, Membrane, Incubation, Flow Cytometry, Expressing, Confocal Microscopy, Imaging, Staining, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Platelets Selectively Regulate the Release of BDNF, But Not That of Its Precursor Protein, proBDNF

doi: 10.3389/fimmu.2020.575607

Figure Lengend Snippet: Molar concentrations of proBDNF are lower in platelets and higher in plasma than those of BDNF. ELISA quantification of proBDNF and BDNF levels in the intraplatelet (A, B) and plasma (C, D) compartments. Concentrations are normalized for 250 x 10 6 platelets. Horizontal bar represents median, *p<0.05. (E, F) Correlation between BDNF and proBDNF molar concentrations in human platelets (E) and in plasma (F) . Dotted lines represent 95% confidence intervals, n=20 participants (#1 to #20).

Article Snippet: Platelets and neuroblastic cells were then incubated at RT with mouse anti-human proBDNF antibody (R&D System, mab31751, monoclonal mouse antibody, clone 584412, diluted 1:25 or Biosensis, R-176 polyclonal rabbit antibody diluted 1:25) or mouse IgG 2b /rabbit isotype control (R&D System, MAB004 and AB-105-C diluted 1:25) for 30 min. Alexa Fluor 488 or 647 conjugated donkey anti-mouse or rabbit secondary antibody (Invitrogen, diluted 1:100) was added for 30 min at RT in the dark.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Platelets Selectively Regulate the Release of BDNF, But Not That of Its Precursor Protein, proBDNF

doi: 10.3389/fimmu.2020.575607

Figure Lengend Snippet: Unlike BDNF, intraplatelet proBDNF is not released during platelet activation. Intraplatelet (A, D) and plasma (B, E) concentrations of BDNF and proBDNF following platelet activation by different agonists. Intraplatelet concentrations are normalized for 250 x 10 6 platelets. Proportion of BDNF (C) and proBDNF (F) in plasma vs . in platelets are expressed in percentage. Error bar represents IQR, *p < 0.05 vs . ctrl, n=20 participants (#1 to #20). Ctrl, control; ADP, adenosine diphosphate; TRAP, thrombin receptor-activating peptide; AA, arachidonic acid.

Article Snippet: Platelets and neuroblastic cells were then incubated at RT with mouse anti-human proBDNF antibody (R&D System, mab31751, monoclonal mouse antibody, clone 584412, diluted 1:25 or Biosensis, R-176 polyclonal rabbit antibody diluted 1:25) or mouse IgG 2b /rabbit isotype control (R&D System, MAB004 and AB-105-C diluted 1:25) for 30 min. Alexa Fluor 488 or 647 conjugated donkey anti-mouse or rabbit secondary antibody (Invitrogen, diluted 1:100) was added for 30 min at RT in the dark.

Techniques: Activation Assay, Clinical Proteomics, Control

Journal: Theranostics

Article Title: A Novel Peptide Interfering with proBDNF-Sortilin Interaction Alleviates Chronic Inflammatory Pain

doi: 10.7150/thno.29703

Figure Lengend Snippet: Mapping of proBDNF-sortilin interaction interface. (A) Schematic diagram illustrating the design of human BDNF prodomain variants. Six variants with a series of amino acids deletions were used in this study. V66M, Val66Met; S, Signal peptide (amino acid 1-18); BDNF, mature BDNF (amino acid 129-247); HA, C-terminal HA epitope. (B) Immunoblots of co-immunoprecipitation (Left) or inputs (Right) of sortilin and proBDNF variants obtained with antibody against HA or Myc epitope. IP, Immunoprecipitation; IB, Immunoblot. (C) Left , live fluorescence micrograph of BiFC mapping assay (Yellow) using HEK293T cells with VC155-sortilin (Sort1) and VN155(I152L)-BDNF prodomain variants. Scale bar represents 50 µm. Right , quantification of fluorescence intensity of BiFC assay. P <0.0001, one-way ANOVA. ** P <0.01 versus Fl group; *** P <0.0001 versus Fl group; ## P <0.001 versus variant I; ### P <0.0001 versus variant I group; n.s., non-significant. Values are means ± SEM. n = 100 individual cells from 4 images. (D) Left , representative immunoblots of supernatant and cell lysates of HEK293 cells overexpressing sortilin and BDNF prodomain variants obtained with antibodies against HA. Right , densitometry analyses of secreted BDNF in supernatants, normalized to input in lysates. P <0.001, one-way ANOVA. * P <0.05, versus Fl group; *** P <0.001, versus Fl group; ## P <0.05, versus I group. Values are means ± SEM. n = 4. IB, Immunoblot. (E) BDNF ELISA quantification of supernatant collected from HEK293 cells overexpressing sortilin and BDNF prodomain variants. P <0.0001, one-way ANOVA. * P <0.05; ** P <0.01; *** P <0.001, versus Sort1/Fl group; ## P <0.05, versus Sort1/variant I group. Values are means ± SEM. Each data point represents the average of 3 independent experiments.

Article Snippet: The primary antibodies used included anti-proBDNF antibody (#ANT-006-AG, Alomone Labs Ltd, Jerusalem, Israel), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (Cell signalling Technology, Danvers, MA, USA), anti-NeuN antibody (MAB377, EMD Millipore Corporation, Billerica, MA, USA) and anti-sortilin antibody (Abcam, Cambridge, UK).

Techniques: Western Blot, Immunoprecipitation, Fluorescence, Mapping Assay, Bimolecular Fluorescence Complementation Assay, Variant Assay, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: A Novel Peptide Interfering with proBDNF-Sortilin Interaction Alleviates Chronic Inflammatory Pain

doi: 10.7150/thno.29703

Figure Lengend Snippet: Construction of a novel peptide targeting proBDNF-sortilin Interaction. (A) Schematic diagram illustrating the design of a series of blocking peptides targeting proBDNF-sortilin interaction interface. Scr-bdnf84-83 (scr), scrambled peptide with permutation of peptide sequence at amino acid 84-93. (B) Immunoblots of co-immunoprecipitation (upper) or reverse co-immunoprecipitation (lower) of sortilin and proBDNF variants in cell lysates overexpressing full-length proBDNF and sortilin followed by peptide incubation obtained with antibodies against HA or Myc. IP, Immunoprecipitation; IB, Immunoblot. (C) Schematic diagram of the design of the Tat-tagged interfering peptide. Scr-bdnf89-98-Tat, Tat-tagged scrambled peptide with permutation of peptide sequence at amino acid 89-98. (D) Live fluorescence micrographs of 5FAM (green) or bright field in rat DRG neuronal cultures before (0 min) and incubated with 5 µM 5FAM-tagged bdnf89-98-Tat (bdnf-Tat) for 2 h. Scale bar represents 150 µm. (E) Fluorescence micrograph of 5FAM (green), sortilin (red) or DAPI (blue) in rat DRG neuronal cultures with peptides pre-treatment for 30 min. Scale bars represent 25 µm. (F) Left , fluorescence micrograph of BiFC assay (yellow) of HEK293T cells overexpressing full-length BDNF and sortilin with vehicle, 10 µM scr-Tat or bdnf-Tat pre-treatment for 30 min. Scale bars represent 25 µm. Right , Determination of the corrected total cell fluorescence (CTCF) of the BiFC signal. P <0.0001 , one-way ANOVA. *** P <0.0001, scr-Tat treatment versus bdnf-Tat treatment. Mean values ± SEM. n = 100 individual cells from 5 images. (G) Effect of 30 min peptide pre-treatment on activity-dependent secretion of BDNF in rat DRG neuronal cultures with high-potassium stimulus using ELISA. P <0.05, one-way ANOVA. * P <0.05, vehicle versus scr-Tat treatment or scr-Tat treatment versus bdnf-Tat treatment. Values are means ± SEM. Each data point represents the average of 3 independent experiments.

Article Snippet: The primary antibodies used included anti-proBDNF antibody (#ANT-006-AG, Alomone Labs Ltd, Jerusalem, Israel), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (Cell signalling Technology, Danvers, MA, USA), anti-NeuN antibody (MAB377, EMD Millipore Corporation, Billerica, MA, USA) and anti-sortilin antibody (Abcam, Cambridge, UK).

Techniques: Blocking Assay, Sequencing, Western Blot, Immunoprecipitation, Incubation, Fluorescence, Bimolecular Fluorescence Complementation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay