bdnf  (OriGene)

Journal: Scientific Reports

Article Title: Human BDNF/TrkB variants impair hippocampal synaptogenesis and associate with neurobehavioural abnormalities

doi: 10.1038/s41598-020-65531-x

Figure Lengend Snippet: Functional characterisation of a rare coding variant in BDNF (E183K). ( A ). Schematic representation of BDNF protein with the common variant (V66M) and rare variant (E183K) indicated. ( B ). PC12 cells were transfected with WT (top)/E183K (bottom) BDNF; neurite length was measured by fluorescence microscopy. Left panel: representative images from 3 experiments. Scale bar: 50 μm. Average neurite length per nucleus is shown (right panel; data point = mean of replicate); *p < 0.05, student’s t-test. ( C ). WT/mutant BDNF was transfected into PC12 cells and protein quantified by Western blot in cell lysate (left) and growth medium (right) using an antibody against a c-terminally fused myc-tag. ( D ). Cultured primary rat hippocampal neurons were co-transfected with RFP-tagged (red) WT BDNF and ClFP-tagged (green) WT BDNF (top image panel), or RFP-tagged WT and ClFP-tagged mutant (E183K) BDNF (bottom image panel). Co-localisation of the proteins was measured by fluorescent confocal microscopy in axons (shown here) and dendrites, and is presented as proportion of vesicles containing both (combined) or only one of the tagged proteins (center panel; data point = one axon). (Right panel: Density of dendritic BDNF positive vesicles containing either WT/WT BDNF or WT/E186K BDNF) Scale bar: 10 μm. ( E ). WT/mutant BDNF expressed in HEK293 cells was immunoprecipitated, followed by Furin-mediated protein cleavage. The cleavage products were analysed by Western blot. ( F ). WT/mutant BDNF were transfected into PC12 cells and depolarisation-dependent BDNF secretion triggered by addition of KCl. Amounts of secreted BDNF were measured by Western blotting. ( G ). TrkB-expressing PC12 cells were transfected with GFP and stimulated with synthetic WT/mutant BDNF. Neurite outgrowth was assessed by fluorescent microscopy (left panel; scale bar: 50 μm); average neurite length per nucleus shown in right panel (data point = mean of replicate). *p < 0.05, student’s t-test. H. Human iPSCs were differentiated into hypothalamic POMC-expressing neurons and stimulated with synthetic WT/mutant BDNF. Neurite outgrowth was assessed by microscopy (left panel; scale bar: 100 μm). Shown are average neurite length per nucleus (right panel; data point = one cell); *p < 0.05, student’s t-test.

Article Snippet: Human cDNA clones of TrkB (RC221794) and BDNF (RC212882) with C-terminal Myc- and Flag-tags were obtained from Origene (Rockville, MD, USA) within the pCMV6-entry backbone.

Techniques: Functional Assay, Variant Assay, Transfection, Fluorescence, Microscopy, Mutagenesis, Western Blot, Cell Culture, Confocal Microscopy, Immunoprecipitation, Expressing

Journal: Scientific Reports

Article Title: Human BDNF/TrkB variants impair hippocampal synaptogenesis and associate with neurobehavioural abnormalities

doi: 10.1038/s41598-020-65531-x

Figure Lengend Snippet: Functional characterisation of TrkB mutants. ( A ). Schematic representation of TrkB protein with rare variants indicated; Immunoglobulin (Ig)-like domain; TM (transmembrane region). ( B ). PC12 cells were transiently transfected with WT/ mutant TrkB. Phosphorylation of downstream signalling molecules was measured by Western Blot before (−) and after (+) stimulation with recombinant BDNF; band intensities quantified by densitometry (right panel; data point = signal from one replicate). Mutants are expressed relative to WT. *p < 0.05, student’s t-test. ( C ). PC12 cells were transiently transfected with WT/mutant TrkB as well as GFP and stimulated with recombinant BDNF for 48 h. Neurite length was measured by fluorescent microscopy (representative images from 2 mutants shown in left panel; remainder shown in Figure ) and quantified as length per nucleus (right panel; data point = mean of one replicate). Scale bar: 50 μm. *p < 0.05, student’s t-test.

Article Snippet: Human cDNA clones of TrkB (RC221794) and BDNF (RC212882) with C-terminal Myc- and Flag-tags were obtained from Origene (Rockville, MD, USA) within the pCMV6-entry backbone.

Techniques: Functional Assay, Transfection, Mutagenesis, Western Blot, Recombinant, Microscopy

Journal: Scientific Reports

Article Title: Human BDNF/TrkB variants impair hippocampal synaptogenesis and associate with neurobehavioural abnormalities

doi: 10.1038/s41598-020-65531-x

Figure Lengend Snippet: Functional characterisation of TrkB mutants in neurons. ( A ). Primary dissociated hippocampal neurons were transfected with WT/mutant TrkB alongside Clover fluorescent protein (ClFP)-tagged actin and stimulated with either vehicle or recombinant BDNF. Dendritic spine density was assessed by confocal microscopy (upper panel: images from one mutant (S714F) shown. Scale bar: 10 μm); lower panel includes data from all mutants (number of spines per 50 μm of dendrite length; data point = one dendrite). *p < 0.01, student’s t-test, ± SEM. ( B ). Patch-clamp experiments were performed on rat hippocampal neurons transiently transfected with WT/mutant TrkB, before (−) and after (+) recombinant BDNF. Shown is the frequency of mEPSCs for all cells recorded. *indicates significant (p < 0.01) increase compared to unstimulated state; by one-way ANOVA-Tukey, F = 28.93. # indicates significant (p < 0.05) decrease compared to unstimulated control (baseline); by one-way ANOVA-Tukey, F = 15.4 ± SEM. ( C ). Shown is amplitude of mEPSCs for all cells recorded. *indicates significant (p < 0.05) reduction compared to BDNF-stimulated control; by one-way ANOVA-Tukey, F = 8.024. # indicates significant (p < 0.05) difference to unstimulated control; by one-way ANOVA-Tukey, F = 10.03. ( D ). Analysis of pre and post-synaptic correlation was done by transfection of ClFP-Actin (to highlight dendritic spines) and WT/mutant TrkB followed by immunostaining of presynaptic terminals with anti-vGlut1 before (−) and after (+) recombinant BDNF addition to DIV11. Representative images are shown in left panel and percentage of dendritic spines (stubby and mushroom) positive for vGlut1 positive presynaptic terminals is shown in right panel ± SEM.

Article Snippet: Human cDNA clones of TrkB (RC221794) and BDNF (RC212882) with C-terminal Myc- and Flag-tags were obtained from Origene (Rockville, MD, USA) within the pCMV6-entry backbone.

Techniques: Functional Assay, Transfection, Mutagenesis, Recombinant, Confocal Microscopy, Patch Clamp, Immunostaining

Journal: Scientific Reports

Article Title: Human BDNF/TrkB variants impair hippocampal synaptogenesis and associate with neurobehavioural abnormalities

doi: 10.1038/s41598-020-65531-x

Figure Lengend Snippet: Effects of TrkB mutants of on dendritic spine maturation. ( A ). To determine the effects of TrkB mutants on dendritic spines size, rat hippocampal neurons - transiently transfected with Clover fluorescent protein-actin and WT/mutant TrkB - were analysed for spine area before (−) and after (+) recombinant BDNF on DIV11. Representative images are shown in Figs. and 4C (images used for quantification). Cumulative frequency plots for control + /− BDNF and mutants + BDNF are shown. In control, stimulation with BDNF (grey vs black), significantly increases the size of spines. p < 2.2*10 −16 , Kolmogorov–Smirnov (K-S) test. Expression of mutant forms of Trkb stunts BDNF’s ability to promote spine growth (colours vs grey). p < 0.001, K-S test. ( B ). To determine the effect of TrkB mutants on post synaptic density sizes, rat hippocampal neurons were transiently transfected with ClFP-Actin and WT/mutant TrkBs (DIV5) and stimulated with vehicle or BDNF (DIV7-11), followed by staining for PSD-95. Representative images are shown (Fig. 4C). Cumulative frequency plots for control + /− BDNF and mutants + BDNF are shown. In control, stimulation with BDNF (grey vs black), significantly increases the area if PSD-95 staining. p < 3*10 −15 , K-S test. Expression of mutant Trkb diminishes BDNF’s stimulatory effect on postsynaptic density size (colours vs grey). p < 2.2*10 −16 , K-S test. ( C ). PSD-95 staining of dendritic spines (mushroom and stubby) is shown in the left-hand panel and proportion of spines positive for PSD-95 staining following expression of WT/mutants is shown in the right-hand panel. PSD-95 staining is reduced for all mutants in the stimulated and unstimulated state. *p < 0.01, student’s t-test ± SEM. ( D ). To determine the effects of TrkB mutants on the surface expression of GluR1-containing AMPA receptors in dendritic spines, rat hippocampal neurons were transiently transfected with ClFP-actin and WT/mutant TrkBs, followed by live staining for GluR1 in the presence or absence of recombinant BDNF (DIV7-11). Representative images are shown (left-hand panel) and the percentage of dendritic spines (mushroom and stubby) positive for surface GluR1 staining is shown in the right-hand panel. In control, stimulation with BDNF significantly (*p < 0.01, student’s t-test ± SEM) increases GluR1 staining in spines, but shows the reverse effect (i.e. decrease) in 3 of the 4 mutants tested ( # p < 0.05, student’s t-test ± SEM).

Article Snippet: Human cDNA clones of TrkB (RC221794) and BDNF (RC212882) with C-terminal Myc- and Flag-tags were obtained from Origene (Rockville, MD, USA) within the pCMV6-entry backbone.

Techniques: Transfection, Mutagenesis, Recombinant, Expressing, Staining