Journal: bioRxiv

Article Title: Cholesterol recognition motifs in the transmembrane domain of the tyrosine kinase receptor family: the case for TRKB

doi: 10.1101/734012

Figure Lengend Snippet: TRKB and cholesterol interaction. (a,b) Snapshots of the interaction between TRKB and cholesterol at the TMR (see supplement video) indicating that cholesterol interacts with the OH- group (red) in Y433 (blue) in TRKB.TMR (green). (c) The interaction between the TRKB CARC motif and cholesterol happens preferentially at the C-alpha in Y433 residues, as indicated by coarse-grained molecular dynamics simulations. (d) The binding of biotinylated BDNF (bBDNF) and TRKB is modulated by cholesterol in a bell-shaped fashion. Red rectangle area is expanded in (e) for the comparison between BDNF 1ng/ml (black circles) with BDNF 0 (ctrl, open circles). Data expressed as Mean/SEM of (c) cholesterol occupancy; or from binding normalized by (d) chol 0/BDNF10ng/ml or (e) chol 0/BDNF 1ng/ml. ∗p<0.05 from control (BDNF 0 or ctrl, C427).

Article Snippet: The plates were washed 3x with PBS buffer, and a mixture of biotinylated BDNF (bBDNF, 0, 0.1, 1 or 10ng/ml, Alomone Labs, #B-250-B) and cholesterol (0, 20, 50 or 100μM) was added for 1h at room temperature, followed by washing with PBS.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

doi: 10.1371/journal.pone.0025097

Figure Lengend Snippet: (A) Role of endogenous BDNF and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF (+), K252a (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.

Article Snippet: Recombinant human BDNF (100 ng/ml), recombinant pro-BDNF (4 ng/ml), and K252a (200 nM) were purchased from Alomone labs.

Techniques: Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

doi: 10.1371/journal.pone.0025097

Figure Lengend Snippet: (A, B) Sortilin as a coreceptor of p75 NTR . Double staining (yellow) of sortilin (red) and p75 NTR (green) in SW480 cells (A) and SW620 (B) after 24 h of serum deprivation. (C) Colocalization of pro-BDNF and sortilin. Confocal microscopy study of a WiDr cells stained with an anti-pro-BDNF Ab (green) and an anti-sortilin Ab (red), and double staining (yellow) after 24 h of serum deprivation. (D, E) apoptotic ratios after 24 h serum deprivation alone (0% FCS) or combined with recombinant Pro-BDNF (0% Pro-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone (0% FCS).

Article Snippet: Recombinant human BDNF (100 ng/ml), recombinant pro-BDNF (4 ng/ml), and K252a (200 nM) were purchased from Alomone labs.

Techniques: Double Staining, Confocal Microscopy, Staining, Recombinant

Journal: Brain

Article Title: Sexual dimorphism in a neuronal mechanism of spinal hyperexcitability across rodent and human models of pathological pain

doi: 10.1093/brain/awab408

Figure Lengend Snippet: NMDARs at female SDH synapses are not potentiated or upregulated by the ex vivo BDNF or in vivo CFA models of pathological pain. ( A ) Baseline lamina I mEPSCs do not differ between male and female adult SD rats. Left : Saline - treated male vs female lamina I neurons, created using BioRender.com. Middle : Average mEPSCs at +60 mV in lamina I neurons from male (green) and female (purple) adult rats. Right : Peak amplitude, decay constant and charge transfer of the NMDAR component of mEPSCs at +60 mV do not differ between male and female SD rats. n = 10 for males and n = 9 for females. Compared using independent samples t -test. ( B ) Male lamina I NMDAR mEPSCs are potentiated following CFA hindpaw injection and ex vivo BDNF treatment. Left : Experimental paradigm showing male in vivo CFA versus ex vivo BDNF models, created using BioRender.com. Middle : NMDAR mEPSCs from male rat lamina I neurons; control in black, CFA in green, BDNF in blue. Right : Charge transfer of NMDAR mEPSCs for groups shown to left . n = 10 for control, n = 8 for CFA and n = 6 for BDNF. Compared using Welch’s test followed by Games–Howell comparison. ( C ) Female lamina I NMDAR mEPSCs are not potentiated following CFA hindpaw injection or ex vivo BDNF treatment. Left : Experimental paradigm showing female in vivo CFA versus ex vivo BDNF models, created using BioRender.com. Middle : NMDAR mEPSCs from female rat lamina I neurons; control in black, CFA in purple, BDNF in blue. Right : Charge transfer of NMDAR mEPSCs shown to left. n = 10 for control, n = 8 for CFA and n = 8 for BDNF. Compared using the Kruskal–Wallis one-way ANOVA. ( D ) Ex vivo BDNF treatment model elicits no effect in female rat SDH synaptosomes. Plots ( left ) and representative western blots ( right ) from female rat SDH synaptosomes of tissue treated with either control saline (lilac, n = 8) or 50 ng/ml recombinant BDNF for 70 min (purple, n = 8; compared using paired-samples t -test). * P < 0.05.

Article Snippet: After tissue preparation, rat or human spinal tissue was placed in oxygenated, room temperature saline containing 50–100 ng/ml recombinant BDNF (Alomone Labs) or control saline for 70 min. A subset of female rat experiments was carried out using an incubation time of 2–4.5 h. Treatment of spinal tissue with BDNF and the kinase inhibitor PP2 (Calbiochem) was carried out using the same approach.

Techniques: Ex Vivo, In Vivo, Injection, Western Blot, Recombinant

Journal: Brain

Article Title: Sexual dimorphism in a neuronal mechanism of spinal hyperexcitability across rodent and human models of pathological pain

doi: 10.1093/brain/awab408

Figure Lengend Snippet: In contrast to males, ex vivo BDNF treatment does not activate markers of disinhibition or facilitated excitation at SDH synapses of female human spinal tissue. ( A ) The ex vivo BDNF model elicits KCC2 internalization in adult male human SDH neurons. Left : Experimental paradigm showing treatment of human SDH tissue in either saline or BDNF, created using BioRender.com. Top right : Average KCC2 intensity values from SDH neurons of male human donor tissue ( n = 12) incubated in saline (light green) versus BDNF (dark green). Bottom right : Comparisons of membrane and intracellular regions by the extra sum-of-squares F -test method; the membrane component is fitted to a Gaussian distribution, while the intracellular component is fitted to an exponential decay. ( B ) The ex vivo BDNF model has no effect on KCC2 internalization in adult female human SDH neurons. Left : Experimental paradigm showing treatment of human SDH tissue in either saline or BDNF, created using BioRender.com. Top right : Average KCC2 intensity values from SDH neurons of saline (lilac) versus BDNF-treated (purple) spinal segments of 10 female human donors. Bottom right: Comparisons of membrane and intracellular regions by the extra sum-of-squares F -test method; the membrane component is fitted to a Gaussian distribution, while the intracellular component is fitted to an exponential decay. ( C and D ) Representative confocal images of male ( C ) and female ( D ) human superficial dorsal horn incubated in saline or BDNF. KCC2 (red), CGRP (green) and NeuN (blue). A zoomed region ( top right ) shows a neuron expressing KCC2 together with the delineation of the membrane and the distance to the membrane of each pixel analysed in a colour-coded distance map. KCC2 intensity (i.u.) versus distance to the membrane profile ( bottom ). Scale bar = 50 µm; inset = 10 µm. ( E ) The ex vivo BDNF treatment model elicits no effect in human female SDH synaptosomes. Plots ( left ) and representative western blots ( right ) from SDH synaptosomes of human female spinal cord treated with either control saline (lilac, n = 6) or 100 ng/ml recombinant BDNF for 70 min (purple, n = 6). Comparisons were made using paired samples t -tests in all cases except for aSTEP 61 , where the Wilcoxon signed-rank test was used. * P < 0.05.

Article Snippet: After tissue preparation, rat or human spinal tissue was placed in oxygenated, room temperature saline containing 50–100 ng/ml recombinant BDNF (Alomone Labs) or control saline for 70 min. A subset of female rat experiments was carried out using an incubation time of 2–4.5 h. Treatment of spinal tissue with BDNF and the kinase inhibitor PP2 (Calbiochem) was carried out using the same approach.

Techniques: Ex Vivo, Incubation, Expressing, Western Blot, Recombinant

Journal: Brain

Article Title: Sexual dimorphism in a neuronal mechanism of spinal hyperexcitability across rodent and human models of pathological pain

doi: 10.1093/brain/awab408

Figure Lengend Snippet: Ovariectomy triggers BDNF-mediated NMDAR potentiation by the KCC2/STEP 61 /SFK pathway in SDH neurons of female rats. ( A ) Ovariectomy (OVX) has no effect on baseline lamina I NMDAR mEPSCs of female SD rats. Left : Saline-treated naïve female versusOVX female lamina I neurons, created using BioRender.com. Middle : Average mEPSCs at +60 mV in lamina I neurons of naïve (purple) and ovariectomized (lilac) female rats. Right : Peak amplitude (compared using Mann–Whitney test), decay constant (compared using independent samples t -test) and charge transfer (compared using independent samples t -test) of the NMDAR component of mEPSCs do not differ between naïve ( n = 10) and OVX ( n = 8) female rats. ( B ) In OVX rats, NMDAR mEPSCs in lamina I neurons are potentiated following ex vivo BDNF treatment. This potentiation is blocked using co-treatment with the SFK inhibitor, PP2. Left : Recordings from lamina I neurons were compared for saline- versus BDNF-treated slices from OVX rats, created using BioRender.com. Middle: Average mEPSCs at +60 mV from OVX female rat lamina I neurons; control in black, BDNF in purple, BDNF+PP2 in blue. Right : Charge transfer of NMDAR mEPSCs shown on left . n = 8 for control, n = 9 for BDNF and BDNF + PP2. Comparisons were made using one-way ANOVA, followed by Tukey HSD when P < 0.05. ( C ) Ex vivo BDNF treatment in OVX rat SDH synaptosomes results in upregulation of pY 420 Fyn and downregulation of KCC2 and STEP 61 . Plots ( left ) and representative western blots ( right ) from OVX female rat SDH synaptosomes of tissue treated with either control saline (lilac, n = 8) or 50 ng/ml recombinant BDNF for 70 min (purple, n = 8, compared using paired samples t -tests). * P < 0.05.

Article Snippet: After tissue preparation, rat or human spinal tissue was placed in oxygenated, room temperature saline containing 50–100 ng/ml recombinant BDNF (Alomone Labs) or control saline for 70 min. A subset of female rat experiments was carried out using an incubation time of 2–4.5 h. Treatment of spinal tissue with BDNF and the kinase inhibitor PP2 (Calbiochem) was carried out using the same approach.

Techniques: MANN-WHITNEY, Ex Vivo, Western Blot, Recombinant