7,8-Dihydroxyflavone elicits TrkB activation in hippocampal neurons. (A) 7,8-Dihydroxyflavone induces TrkB tyrosine phosphorylation in hippocampal neurons. Immunofluorescent staining was conducted with anti-phospho-TrkB Y816. (B) Immunoblotting shows 7,8-dihydroxyflavone triggers TrkB phosphorylation in hippocampal neurons. (C) K252a blocks 7,8-dihydroxyflavone’s agonistic effect on TrkB. Hippocampal neurons were pretreated with K252a (30 nM) for 30 min, followed by BDNF (100 ng/mL) or 7,8-dihydroxyflavone (500 nM) for 30 min. Cell lysates were analyzed by immunoblotting. (D) 7,8-Dihydroxyflavone stimulates Akt and ERK phosphorylation. (E) 7,8-Dihydroxyflavone induces TrkB phosphorylation in cortex of BDNF conditional knockout mice. Mice were injected i.p. with 5 mg/kg 7,8-dihydroxyflavone or vehicle. After 2 h, the mice were killed, and cortex lysates were analyzed by immunoblotting. (F) 7,8-dihydroxyflavone induces TrkB phosphorylation in cortex of BDNF conditional knockout mice. Mice were intraperitoneally injected with 5 mg/kg 7,8-dihydroxyflavone or vehicle. After 2 h, mouse cortex lysates were analyzed by immunoblotting.

7,8-Dihydroxyflavone is neuroprotective in models of neuronal injury. (A ) 7,8-Dihydroxyflavone decreases KA-induced apoptosis in mouse brain in a TrkB-dependent manner. TrkB F616A mice were injected with various indicated solutions, and the brain slides were analyzed with TUNEL assay. Results are expressed as mean ± SEM (ANOVA; n = four to five mice/group; #, P < 0.005; *, P < 0.01; **, P < 0.1;). (B) 7,8-Dihydroxyflavone diminishes stroke damage in a TrkB-dependent manner. Infarct volumes after 24 h MCAO were substantially decreased by 7,8-dihydroxyflavone. Pretreatment with 1NMPP1 impaired the protective effect of 7,8-dihydroxyflavone. Results are expressed as mean ± SEM (One way ANOVA, n = 8–12 mice/group; #, not significant; *, P < 0.01; **, P < 0.02). (C) 7,8-Dihydroflavone is neuroprotective in a model of Parkinson disease. Mice were administered 7,8-dihydroxyflavone in drinking water for 14 days. On day 7, the mice were given two doses of MPTP (20 mg/kg, i.p.) 2 h apart. On day 14, mice were killed. Immunostaining (substantia nigra, striatum) (Upper panels) and immunoblotting (striatal homogenates, lanes from the same gel at the same exposure) of tyrosine hydroxylase (TH) (Lower panels), and fluorescence microscopy of activated caspase-3 in TH+ nigral neurons revealed reduced toxicity in 7,8-dihydroxyflavone-treated mice (Lower panels). (D) 7,8-Dihydroxyflavone prevents glutamate-triggered neuronal apoptosis in wild-type but not TrkB-null neurons. Cortical neurons were prepared from the P0 pups. The neurons were pretreated with indicated compounds, followed by glutamate (50 μM). The cell lysates were analyzed by immunoblotting. (E) 7,8-Dihydroxyflavone selectively activates TrkB F616A, which can be blocked by 1NMPP1. The primary cortical cultures were pretreated for 30 min with either K252a (100 nM) or 1NMPP1 inhibitor (100 nM), followed by 7,8-dihydroxyflavone. Immunoblotting with cell lysates was performed. (F) 7,8-Dihydroxyflavone suppresses KA-induced neuronal cell death in TrkB F616A mutant mice, which can be blocked by 1NMPP1.

7,8-Dihydroxyflavone protects hippocampal neurons from apoptosis. (A) Chemical structures of flavones. (B) Flavone derivatives prevent apoptosis in T48 but not SN56 cells. (C) Titration of the EC₅₀ for suppressing apoptosis in T48 cells. (D) 7,8-Dihydroxyflavone protects hippocampal neurons from glutamate-triggered apoptosis. Hippocampal neurons were pretreated with 500 nM flavone derivatives for 30 min, followed by 50 μM glutamate for 16 h. The cell lysates were analyzed by immunoblotting. (E) 7,8-Dihydroxyflavone protects rodent neurons from apoptosis triggered by OGD. Hippocampal neurons were seeded on coverslips in a 24-well plate. The neurons were pretreated with various flavones for 30 min followed by OGD at 37 °C for 3 h. Neuronal apoptosis was quantitatively analyzed (Left). 7,8-Dihydroxyflavone displayed a dose-dependent protection on neurons (Right). Data are expressed as mean ± SEM.

7,8-Dihydroxyflavone binds ECD of TrkB and provokes its dimerization and phosphorylation. (A) 7,8-Dihydroxyflavone provokes TrkB dimerization. mGST-TrkB and HA-TrkA or HA-TrkB were cotransfected into HEK293 cells and treated with 0.5 μM pinocembrin or 7,8-dihydroxyflavone for 30 min. GST-TrkB was pulled down and analyzed with anti-HA-HRP. (B) 7,8-Dihydroxyflavone induces TrkB autophosphorylation. (C) [3H]7,8-dihydroxyflavone binds the ECD but not ICD of TrkB. In vitro binding assay with purified TrkB ECD or ICD and [3H]7,8-dihydroxyflavone (Upper). Scatchard plot analysis of 7,8-dihydroxyflavone binding to TrkB (Lower). (D) In vitro binding assay with immobilized GST-TrkB ECD or ICD and 7,8-dihydroxyflavone. Gradual increments of GST-TrkB ECD but not ICD decreased 7,8-dihydroxyflavone in the eluted fractions. (E) Mapping assay with various ECD truncates and [3H]7,8-dihydroxyflavone. Data are expressed as mean ± SEM.