Journal: Nature cell biology

Article Title: STAT3-BDNF-TrkB signaling promotes alveolar epithelial regeneration after lung injury

doi: 10.1038/s41556-020-0569-x

Figure Lengend Snippet: (A) AT2 cells were isolated from an uninjured C57BL/6 mouse and from a C57BL/6 mouse that underwent acid-induced lung injury 24 hours prior. Single-cell transcriptomic analysis was performed and cells were grouped into populations using t-SNE. There was little overlap between AT2 cells from uninjured and injured mice. (B) K-means analysis was used to sub-divide AT2 cells into groups based on their individual gene expression. (C) Genes whose expression was unique to each cluster are shown. Cluster 3 has gene expression from both AT2 and AT1 cells (Sftpc and Pdpn) and AEP marker Tm4sf1. (D) Bdnf is the only bona fide STAT3 target gene with newly accessible chromatin following acid-induced lung injury that is differentially expressed in cluster 3. (E) Data from ATAC-seq (shown in Fig. 1) demonstrate increased accessibility of the Bdnf promotor in cells isolated from mice 24 hours after acid-induced lung injury. (F) AT2 cells from tamoxifen-exposed SftpcCreERT2:Stat3LoxP/LoxP mice express less Bdnf than vehicle exposed mice (n=4 mice per group) (G) BAL fluid from SftpcCreERT2:Stat3LoxP/LoxP mice treated with either tamoxifen or corn oil (vehicle) prior to undergoing acid-induced lung injury shows that mice lacking AT2-specific Stat3 have significantly less BDNF in their BAL fluid (n=3 mice per group per time point). (H) AT2 cells from SftpcCreERT2-Rosa26TdTomato:Hopx3FlagGFP mice were co-cultured with PDGFRα+ mesenchymal cells from C57BL/6 mice in the presence of BDNF which increased alveolar organoid forming efficiency of murine AT2 cells. Right-hand panels show quantification from n=3 distinct cultures per condition. (I) Primary human AT2 cells were isolated and co-cultured with MRC5 fibroblasts for three weeks. We observed increased organoid size and forming efficiency when BDNF was added to the culture media. Right-hand panels show the average organoid forming efficiency and size from n=3 different donors. Individual points are shown with the mean and standard error of the mean depicted. For panels (F), (G), (H), and (I), data is shown as the mean +/− SEM. Statistical significance was determined with a two-tailed Student’s t-test (F), (H), and (I), and with a two-way ANOVA (G). Scale bars, 1mm.

Article Snippet: After two days of culture, Y276632 was removed and ligand treatments of organoids were performed using the following reagents at the indicated concentrations: ANA-12 0.02μg/mL (Alomone Labs catalog #A-215), recombinant human BDNF 0.05μg/mL (Alomone Labs catalog #B-250) and recombinant murine FGF7 0.01μg/mL (R&D Systems catalog #5028-KG).

Techniques: Isolation, Single Cell, Analysis, Gene Expression, Expressing, Marker, Cell Culture, Two Tailed Test, Single Cell RNA-Seq

Journal: Nature cell biology

Article Title: STAT3-BDNF-TrkB signaling promotes alveolar epithelial regeneration after lung injury

doi: 10.1038/s41556-020-0569-x

Figure Lengend Snippet: (A) We quantified tomato positive AT2 cells in BdnfCre-R26TdTomao mice before and 24h after acid-induced lung injury and found a significant increase in co-positive cells following acute lung injury (n=4 mice per group). (B-F) H&E staining and ATS lung injury scores (B), BAL BDNF concentrations (C) BAL protein concentration (D) AT2 cell numbers (E) and number of proliferating AT2 cells (F) of tamoxifen-exposed and vehicle-exposed SftpcCreERT2:BdnfLoxP/LoxP mice 5 days after acid-included lung injury (n= 3 mice per time point for C & D, n=4 mice per group per time point for B, E, & F). (G) H&E and PDPN staining of lung tissue from tamoxifen-exposed and vehicle-exposed SftpcCreERT2:BdnfLoxP/LoxP mice that had been infected with intranasal PR8 influenza (5x10−5 HAU/mouse) (4 mice per group). (H) KRT5 staining of the mice described in (G) with quantification of KRT5+ pods in both groups (n=4 mice per group). For panels (A), (B), (C), (D), (E), (F) and (H), data is shown as the mean +/− SEM. Statistical significance was determined with a two-tailed Student’s t-test (A), (B), (E), (F), and (H), and with a two-way ANOVA (C) and (D). Scale bars, 100μm (B, G, H) and 10μm (A).

Article Snippet: After two days of culture, Y276632 was removed and ligand treatments of organoids were performed using the following reagents at the indicated concentrations: ANA-12 0.02μg/mL (Alomone Labs catalog #A-215), recombinant human BDNF 0.05μg/mL (Alomone Labs catalog #B-250) and recombinant murine FGF7 0.01μg/mL (R&D Systems catalog #5028-KG).

Techniques: Staining, Protein Concentration, Tissue, Infection, Two Tailed Test, Immunofluorescence

Journal: Nature cell biology

Article Title: STAT3-BDNF-TrkB signaling promotes alveolar epithelial regeneration after lung injury

doi: 10.1038/s41556-020-0569-x

Figure Lengend Snippet: BdnfCre-Rosa26tdTomato were subjected to acid-induced lung injury (n=4 mice per group). One day after injury the mice were euthanized, and the lungs were probed for Tomato (red) and proSFTPC (green). Autofluorescence delineates tissue structure. Scale bar, 25μm.

Article Snippet: After two days of culture, Y276632 was removed and ligand treatments of organoids were performed using the following reagents at the indicated concentrations: ANA-12 0.02μg/mL (Alomone Labs catalog #A-215), recombinant human BDNF 0.05μg/mL (Alomone Labs catalog #B-250) and recombinant murine FGF7 0.01μg/mL (R&D Systems catalog #5028-KG).

Techniques: Tissue, Immunofluorescence

Journal: Nature cell biology

Article Title: STAT3-BDNF-TrkB signaling promotes alveolar epithelial regeneration after lung injury

doi: 10.1038/s41556-020-0569-x

Figure Lengend Snippet: (A) Fgf7 mRNA expression in mesenchymal cells isolated from the organoid culture conditions described in Fig. 4H (n= 5 wells per condition). (B) Fgf7 expression in PDFRα cells isolated from tamoxifen or corn oil exposed SftpcCreERT2:Stat3LoxP/LoxP, SftpcCreERT2:BdnfLoxP/LoxP, and PdgfraCreERT2:TrkBLoxP/LoxP mice 24 hours after acid-induced lung injury (n=4 mice per group). (C) The addition of 0.1μg/mL of recombinant murine BDNF (rmBDNF) caused a significant increase in alveolar organoid forming efficiency and size (n=3 wells per condition). (D) In the setting of acute lung injury STAT3-activating cytokines accumulate in the alveolus and STAT3 becomes activated in the AT2 cell. Accompanying changes in chromatin accessibility within AT2 cells allows for expression of BDNF in transdifferentiating cells which then binds to TrkB on mesenchymal niche cells to stimulate expression of Fgf7, which promotes alveolar epithelial regeneration. For panels (A) (B) and (C), data is shown as the mean +/− SEM. Statistical significance was determined with a two-tailed Student’s t-test (A) (B) and (C). Scale bars, 500μm.

Article Snippet: After two days of culture, Y276632 was removed and ligand treatments of organoids were performed using the following reagents at the indicated concentrations: ANA-12 0.02μg/mL (Alomone Labs catalog #A-215), recombinant human BDNF 0.05μg/mL (Alomone Labs catalog #B-250) and recombinant murine FGF7 0.01μg/mL (R&D Systems catalog #5028-KG).

Techniques: Expressing, Isolation, Recombinant, Two Tailed Test

Journal: Antioxidants (Basel, Switzerland)

Article Title: A Novel Gastrodin Derivative with Neuroprotection Promotes NGF-Mimic Activity by Targeting INSR and ACTN4 to Activate PI3K/Akt Signaling Pathway in PC12 Cells.

doi: 10.3390/antiox14030344

Figure Lengend Snippet: Figure 3. Screening NGF-mimic activity of GAD037-related potential targets and signaling pathways. (A–I) Effects of inhibitors of TrkA, TrkB, Ras, Raf, ERK, GR, IGF-1R, INSR, PI3K (K252a, ANA-12, FTA, AZ628, U0126, RU486, T9576, HNMPA-(AM)3, and LY294002, respectively) on neurite outgrowth in- duced by GAD037 treatment for 48 h. All experiments with inhibitors were conducted simultaneously in 3rd, 4th, and 9th generations of cells. Data are presented as mean ± SEM. *** p < 0.001 represents significant differences compared with control group; ns indicate no significant differences; # and ### indicate significant differences at p < 0.05 and p < 0.001, respectively.

Article Snippet: The pronase E (CAT No.: HY-114158) was bought from MedChemExpress, Shanghai, China, while tyrosine kinase B (TrkB) inhibitor (ANA-12, CAT No.: S7745) was acquired from Selleck, Shanghai, China.

Techniques: Activity Assay, Protein-Protein interactions, Control

Journal: Nature Communications

Article Title: BDNF rescues BAF53b-dependent synaptic plasticity and cocaine-associated memory in the nucleus accumbens

doi: 10.1038/ncomms11725

Figure Lengend Snippet: ( a ) Bath application of 500 nM ANA12 blocked theta burst-induced LTP (TBS 1 ) in NAc ( n =6) in slices from WT mice. A 60-min period of ANA12 washout restored LTP after a second bout of theta stimulation (TBS 2 ) in the same stimulated pathway. Inset, representative traces collected during baseline (grey line), 60 min post-TBS 1 (blue line) and 60 min post-TBS 2 (dark blue line). Scale bar, 0.4 mV, 5 ms. ( b ) Infusion of BDNF (2 nM) began after a 15-min stable baseline recording. BDNF produced a rapid increase in glutamatergic transmission (∼35%) within the NAc in slices from both WT ( n =6) and Baf53bΔHD ( n =5) mice. Before delivery of theta bursts, stimulation intensity was reduced to reset baseline to pre-drug levels, to evaluate the effects of BDNF on the magnitude of LTP (dotted downward arrow). Five theta trains (five theta bursts each, separated by 1 min) delivered 5 min after re-establishing stable baseline recordings (upward arrow) produced robust and stable LTP in WT slices and completely reversed the LTP impairment previously seen in slices from Baf53bΔHD mice (see ). Inset, representative traces collected during baseline (grey line), 30 min after start of BDNF infusion (blue line) and 60 min post TBS (dark blue line). Scale bar. 0.4 mV, 5 ms. ( c ) Pretreatment with 500 nM ANA12 completely blocked BDNF-induced increases in baseline synaptic transmission. The delivery of TBS produced a brief short-term potentiation that rapidly returned towards baseline levels ( n =6). Inset, representative traces collected during baseline (grey line) and 60 min post TBS (black line). Scale bar, 0.5 mV, 5 ms.

Article Snippet: The AMPA receptor antagonist, DNQX (Tocris) and human recombinant form of BDNF (Chemicon) were prepared fresh in aCSF, whereas the TrkB receptor antagonist, ANA12 (Tocris), stock was dissolved in dimethyl sulfoxide and then diluted to working concentration in aCSF (dimethyl sulfoxide <0.01%).

Techniques: Produced, Transmission Assay

Journal: Scientific Reports

Article Title: Blockade of soluble epoxide hydrolase attenuates post-ischemic neuronal hyperexcitation and confers resilience against stroke with TrkB activation

doi: 10.1038/s41598-017-18558-6

Figure Lengend Snippet: The functional relevance of TrkB overactivation in the sEH knockout (sEH KO) mice evaluated by pretreatment with the selective TrkB antagonist ANA12 before oxygen-glucose deprivation (OGD) ex vivo and before middle cerebral artery occlusion (MCAO) in vivo . ( A ) Representative traces of the field excitatory postsynaptic potentials (fEPSPs) in the hippocampus at different time points at baseline, during OGD induction and during post-ischemic long-term potentiation (iLTP) expression. The averaged time course of fEPSP changes over 60 minutes following OGD induction is shown for WT and sEH KO mice with either vehicle or ANA12 pretreatment. ( B ) During iLTP expression in the last 10 minutes, the average fEPSP amplitude significantly decreased in the sEH KO/vehicle group relative to the WT/vehicle group; this effect was abolished by ANA12 pretreatment in the sEH KO group (sEH KO/ANA12, n = 6/group). ( C ) Vital staining of representative brain slices at 48 hours after MCAO from WT and sEH KO mice with either vehicle or ANA12 pretreatment. ( D ) ANA12 pretreatment (sEH KO/ANA12, n = 6) significantly eliminated the infarct reduction in the sEH KO/vehicle relative to the WT/vehicle (n = 6/group), suggesting that the protective effects of sEH deletion on the attenuation of iLTP and infarction were mediated by TrkB activation. ( E ) A mechanistic framework summarizing the effects of sEH blockade. Blocking sEH induces TrkB overactivation and prevents EET degradation, leading to neuroprotection, iLTP reduction, vasodilatation, and, finally, reductions in infarcts and their associated deficits. Calibration: 0.2 mV, 5 ms. The values represent the mean ± standard error of the mean. *, # p < 0.05, ***, ### p < 0.001.

Article Snippet: WT and sEH KO mice were pretreated with intraperitoneal injection of ANA12 (0.5 mg/kg, MedKoo; n = 5/group) or vehicle (1% DMSO, Sigma-Aldrich; n = 3/group) every 12 hours for 3 times before the MCAO surgery to ensure TrkB inhibition.

Techniques: Functional Assay, Knock-Out, Ex Vivo, In Vivo, Expressing, Staining, Activation Assay, Blocking Assay