Journal: Journal of Clinical Investigation

Article Title: Small molecule BDNF mimetics activate TrkB signaling and prevent neuronal degeneration in rodents

doi: 10.1172/jci41356

Figure Lengend Snippet: Figure 3 LM22A compounds function through TrkB. (A) Survival analysis of hippocampal neurons treated with BDNF (0.7 nM) or LM22A compounds (500 nM) with or without the Trk inhibitor K252a (200 nM). For BDNF, n = 37 wells derived from 7 experiments; for LM22A-1, -2, and -3, n = 17–21 wells derived from 6 experiments; and for LM22A-4, n = 28–57 wells derived from 4 experiments were assessed. (B) Survival analysis of hippocampal neurons treated with CM, BDNF (0.7 nM), or LM22A compounds (500 nM) with or without TrkBECD antibody. For each condition, n = 37–42 wells derived from 5 experiments were assessed. (C) TUNEL/DAPI analysis of E16 hippocampal neurons treated with CM, BDNF (0.7 nM), or LM22A compounds (500 nM) with or without TrkBECD antibody. For each condition, n = 17–31 fields derived from 3 experiments were assessed. (D) LM22A compounds or neurotrophic factors were incubated for 60 minutes with NIH-3T3 cells stably expressing specific Trk receptors. Western blot analysis (anti–pan-phospho-TrkY490 [p-Trk]) demonstrated that LM22A compounds activated TrkB (upper panels) but not TrkA (middle panels) or TrkC (lower panels), while all 3 Trks were activated by their cognate ligands. Activa- tion patterns of 3 additional independent assays were identical. (E–H) Trk- and p75NTR-specific NIH-3T3 cells were incubated in serum-free medium in the presence of the designated ligands for 72–96 hours, and survival was measured using the EnzyLight assay. n = 18–28 wells derived from 11–14 experiments. *P < 0.05, **P < 0.01, §P < 0.001.

Article Snippet: Other sources include the following: LY294002, K252a, and PD98059, Calbiochem; mouse monoclonal anti–phospho-ERKT202/Y204, rabbit polyclonal anti-ERK42/44, mouse monoclonal anti–phosphoAKTS473, rabbit polyclonal anti-AKT, and site-specific rabbit polyclonal anti-TrkY490, Cell Signaling Technology Inc. Antibodies were obtained from the following sources: mouse monoclonal anti-BDNF, Sigma- Aldrich; rabbit polyclonal TrkA and TrkB antibodies, Upstate USA Inc.; mouse monoclonal TrkB antibody, BD Transduction Laboratories; TrkC rabbit polyclonal antibody, Santa Cruz Biotechnology Inc.; rabbit DARPP32 polyclonal antibody, Millipore; recombinant human NGF, Invitrogen; recombinant human BDNF, Sigma-Aldrich; recombinant chimeric proteins ephrin-A5-Fc and Trk-Fc, R&D Systems.

Techniques: Derivative Assay, TUNEL Assay, Incubation, Stable Transfection, Expressing, Western Blot

Journal: Journal of Clinical Investigation

Article Title: Small molecule BDNF mimetics activate TrkB signaling and prevent neuronal degeneration in rodents

doi: 10.1172/jci41356

Figure Lengend Snippet: Figure 7 LM22A-4 inhibits neuronal death in in vitro neurodegenerative disease models. (A) Six- to 7-DIV hippocampal neurons from E16 mice were treated in the absence of Aβ with CM alone or CM + K252a (K) or in the presence of oligomeric Aβ with CM alone, CM + K252a, BDNF (0.7 nM), BDNF + K252a, LM22A-4 (500 nM), LM22A-4 + K252a. K252a was used at 200 nM. After 72 hours, cultures were assessed by TUNEL/DAPI staining. n = 28–29 fields for each condition derived from a total of 3 experiments. (B) SH-SY5Y human neuroblastoma cells were pretreated for 3 days in the following conditions: CM alone, CM + K252a, BDNF (0.7 nM), BDNF + K252a, LM22A-4 (500 nM), LM22A-4 + K252a. MPP+ (100 μM) was added, and 48 hours later cell survival was assessed by MTT/cell count assay. (C) Six- to 7-DIV striatal neurons from E16 mice were pretreat- ed for 2 hours in the following conditions: CM, CM + K252a, BDNF (0.7 nM), BDNF + K252a, LM22A-4 (500 nM), LM22A-4 + K252a. QA (7.5 mM) was added, and after 24 hours numbers of DARPP-32–positive/total surviving cells was determined. n = 50–100 fields for each condition derived from a total 5 experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Other sources include the following: LY294002, K252a, and PD98059, Calbiochem; mouse monoclonal anti–phospho-ERKT202/Y204, rabbit polyclonal anti-ERK42/44, mouse monoclonal anti–phosphoAKTS473, rabbit polyclonal anti-AKT, and site-specific rabbit polyclonal anti-TrkY490, Cell Signaling Technology Inc. Antibodies were obtained from the following sources: mouse monoclonal anti-BDNF, Sigma- Aldrich; rabbit polyclonal TrkA and TrkB antibodies, Upstate USA Inc.; mouse monoclonal TrkB antibody, BD Transduction Laboratories; TrkC rabbit polyclonal antibody, Santa Cruz Biotechnology Inc.; rabbit DARPP32 polyclonal antibody, Millipore; recombinant human NGF, Invitrogen; recombinant human BDNF, Sigma-Aldrich; recombinant chimeric proteins ephrin-A5-Fc and Trk-Fc, R&D Systems.

Techniques: In Vitro, TUNEL Assay, Staining, Derivative Assay, Cell Counting

Journal:

Article Title: Actions of brain-derived neurotrophic factor on evoked and spontaneous EPSCs dissociate with maturation of neurones cultured from rat visual cortex

doi: 10.1111/j.1469-7793.2000.t01-1-00579.x

Figure Lengend Snippet: A, to the left is shown the time course of the frequency of mEPSCs. The frequency was measured every 30 s. BDNF and K252a were applied to this cell at 200 ng ml−1 and 200 nm, respectively, during the periods indicated by horizontal bars. To the right are shown cumulative plots of the amplitude distributions of mEPSCs 2–5 min before (continuous line) and 17–20 min after (dotted line) the BDNF application. Bin width is 2 pA. The total number of events before and after the BDNF application is 572 and 587, respectively. Other conventions are the same as in Fig. 1D. B, to the left is shown the time course of the frequency of mEPSCs. BDNF and anti-BDNF antibody were applied to this cell at 200 ng ml−1 and 5 μg ml−1, respectively, during the periods indicated by the horizontal bars. To the right are shown cumulative plots of the amplitude distributions of mEPSCs 2–5 min before (continuous line) and 17–20 min after (dotted line) the BDNF application. The total number of events is 452 and 464, respectively. Other conventions are the same as in Fig. 1D.

Article Snippet: Application of BDNF and drugs In most of the experiments, recombinant human BDNF (provided from Sumitomo Pharmaceutical Co., Ltd, Japan), or K252a (Kyowa Hakko Co., Ltd, Japan) was applied to neurones through the perfusion medium 10–15 min after the initiation of recordings.

Techniques:

Journal:

Article Title: Actions of brain-derived neurotrophic factor on evoked and spontaneous EPSCs dissociate with maturation of neurones cultured from rat visual cortex

doi: 10.1111/j.1469-7793.2000.t01-1-00579.x

Figure Lengend Snippet: Ratio of the frequency of mEPSCs during the application of BDNF alone, BDNF + K252a and BDNF + anti-BDNF antibody to the respective control value at the time points shown on the abscissa. Filled symbols with vertical bars represent means and twice the s.d. for the same group of cells.

Article Snippet: Application of BDNF and drugs In most of the experiments, recombinant human BDNF (provided from Sumitomo Pharmaceutical Co., Ltd, Japan), or K252a (Kyowa Hakko Co., Ltd, Japan) was applied to neurones through the perfusion medium 10–15 min after the initiation of recordings.

Techniques:

Journal: Antioxidants

Article Title: 3,3′-Diindolylmethane Promotes BDNF and Antioxidant Enzyme Formation via TrkB/Akt Pathway Activation for Neuroprotection against Oxidative Stress-Induced Apoptosis in Hippocampal Neuronal Cells

doi: 10.3390/antiox9010003

Figure Lengend Snippet: Inhibitory effect of K252a or MK-2206 on neuroprotective action of DIM. HT-22 cells were preincubated with or without DIM in combination with K252a or MK-2206 for 30 min before glutamate challenge. After 12 h, cell viability and ROS level were measured as described in Materials and Methods. ( A ), ( B ) Cell viability and ( C ), ( D ) ROS level. Data are the mean ± SD values of quintuple determinations. ** p < 0.01 versus glutamate-treated group; # p < 0.05 and ## p < 0.01 versus DIM with glutamate-treated group.

Article Snippet: DIM, K252a (a TrkB inhibitor), and MK-2206 (a specific Akt inhibitor) were procured from Cayman Chemical Company (Ann Arbor, MI, USA).

Techniques: