Flavonoid fisetin promotes ERK-dependent long-term potentiation and enhances memory

Abstract

Small molecules that activate signaling pathways used by neurotrophic factors could be useful for treating CNS disorders. Here we show that the flavonoid fisetin activates ERK and induces cAMP response element-binding protein (CREB) phosphorylation in rat hippocampal slices, facilitates long-term potentiation in rat hippocampal slices, and enhances object recognition in mice. Together, these data demonstrate that the natural product fisetin can facilitate long-term memory, and therefore it may be useful for treating patients with memory disorders.

Fig. 1.

Structure of the flavonoid fisetin.

Fig. 2.

Fisetin activates ERK1 (p44), ERK2 (p42), and CREB in rat hippocampal slices. (A) Hippocampal slices in ACSF were treated with 1 μM fisetin for 5–20 min, and then equal amounts of protein were analyzed by SDS/PAGE and immunoblotting with antibodies to phospho-ERK and phospho-CREB along with antibodies to the unphosphorylated forms of the proteins, demonstrating no changes in overall protein levels. Similar results were obtained in two independent experiments. (B) Hippocampal slices were pretreated for 30 min with either 50 μM PD98059 (PD) or 10 μM U0126 (U) before the addition of 1 μM fisetin for 5 min. Samples were analyzed as in A. (C) The average phosphoprotein signal from the blots in A, quantified by densitometry and normalized to total protein, was plotted ±SD. The asterisks indicate a significant difference from the control (P < 0.005). (p42: 5 min, 1.68 ± 0.06; 10 min, 1.31 ± 0.05; 20 min, 1.42 ± 0.03; p44: 5 min, 1.98 ± 0.16; 10 min, 1.48 ± 0.02; 20 min, 1.30 ± 0.09; CREB: 5 min, 2.87 ± 0.56; 10 min, 2.72 ± 0.12; 20 min, 2.55 ± 0.39.) (D) The average phosphoprotein signal from the blots in B, quantified by densitometry and normalized to total protein, was plotted ±SD. ∗, significant difference from the control (P < 0.0005).#, significant difference from fisetin alone (P < 0.0001). (p42: PD98059, 0.83 ± 0.08; U0126, 0.30 ± 0.05; 5 min fisetin, 1.68 ± 0.06; fisetin + PD98059, 0.72 ± 0.09; fisetin + U0126, 0.54 ± 0.06; p44: PD98059, 0.75 ± 0.05; U0126, 0.13 ± 0.06; 5 min fisetin, 1.98 ± 0.16; fisetin + PD98059, 0.70 ± 0.10; fisetin + U0126, 0.29 ± 0.05; CREB: PD98059, 0.64 ± 0.11; U0126, 0.71 ± 0.31; 5 min fisetin, 2.87 ± 0.56; fisetin + PD98059, 0.55 ± 0.05; fisetin + U0126, 0.20 ± 0.03.) Similar results were obtained in two independent experiments.

Fig. 3.

Fisetin facilitates the induction of LTP in Schaffer collateral CA1 pyrimidal cell synapses in rat hippocampal slices. (A) Effect of fisetin (1 μM, n= 6) on basal synaptic transmission. Hippocampal slices were exposed to fisetin during the time indicated by the black bar. The field excitatory postsynaptic potential (fEPSP) slope is expressed as the percentage of the value immediately before the addition of fisetin. (A Insets) Representative records 5 min before (Inset 1) and 60 min after (Inset 2) exposure to fisetin. Fisetin does not affect basal synaptic transmission. (B and C) Fisetin facilitates the induction of LTP after a weak tetanic stimulation (15 pulses at 100 Hz), which alone does not induce LTP in control slices. The effect of fisetin is dose-dependent. The hippocampal slices were untreated (n= 14) or exposed to fisetin (0.1 μM, n= 7; 1 μM, n= 8; 5 μM, n= 6; 10 μM, n= 6) for the time indicated by the black bar, and weak tetanic stimulation was applied at time 0. The fEPSP slope is expressed as the percentage of the value immediately before the application of weak tetanic stimulation. (B) Time course of changes in the fEPSP slope. (B Insets) Representative records at −25 min (Inset 1) and + 60 min (Inset 2) in control and 1 μM fisetin-treated slices. To compare the data among the groups, the averages of the fEPSP slopes 30–60 min after tetanic stimulation were calculated as an index of LTP magnitude; they are shown in C. (C) None: 108.0 ± 3.4%; 0.1 μM fisetin: 124.8 ± 14.6%, not significant vs. none; 1 μM fisetin: 150.1 ± 16.3%, **, P < 0.01 vs. none; 5 μM fisetin: 146.9 ± 7.9%, *, P < 0.05 vs. none; 10 μM fisetin: 150.1 ± 16.3%, ∗∗, P < 0.01 vs. none; 5 μM fisetin: 146.9 ± 7.9%, ∗, P < 0.05 vs. none; 10 μM fisetin: 125.2 ± 6.5%, not significant vs. none (ANOVA followed by Dunnett's test). All data are the mean ± SEM. (D) The facilitation of LTP by fisetin depends on the timing of its addition relative to the tetanic stimulus. Fisetin must be present during the stimulus to show facilitation of LTP. The hippocampal slices were untreated (n= 9) or exposed to 1 μM fisetin before (filled bar, n= 5) or after (open bar, n= 5) application of weak tetanic stimulation (15 pulses at 100 Hz). Data are presented as in B.

Fig. 4.

Involvement of the MEK/ERK cascade in fisetin-induced facilitation of LTP in rat hippocampal slices. (A) Effect of fisetin (1 μM, n= 6) on NMDA receptor-mediated fEPSP in Mg²⁺-free medium. The hippocampal slices were exposed to fisetin (black bar) and the NMDA receptor antagonist 2-amino-5-phosphonovalerate (APV, white bar). The NMDA receptor-mediated fEPSP area is expressed as the percentage of the value immediately before addition of fisetin. (A Insets) Representative records 5 min before (Inset 1) and 25 min after (Inset 2) exposure to fisetin (Inset 2) and 10 min after exposure to APV (Inset 3). Fisetin has no effect on NMDA receptor-mediated synaptic responses. (B and C) MEK inhibitors PD98059 and U0126 block fisetin-dependent facilitation of LTP. The hippocampal slices were untreated (n= 12) or exposed to 1 μM fisetin alone (n= 7) or to a weak tetanic stimulation applied at time 0 after a 10-min pretreatment with PD98059 (50 μM, n= 6) or U0126 (20 μM, n= 5). (B) Time course of changes in the fEPSP slope. To compare the data among the groups, the averages of the fEPSP slopes 30–60 min after tetanic stimulation were calculated as an index of LTP magnitude and are shown in C. (C) None: 104.5 ± 4.0%; fisetin: 143.9 ± 11.4%, ∗∗, P < 0.01 vs. none; fisetin + PD98059: 103.9 ± 5.5%, , P < 0.05 vs. fisetin; fisetin + U0126: 104.7 ± 8.3%, , P < 0.05 vs. fisetin (ANOVA followed by Tukey–Kramer test). All data are mean ± SEM. (D and E) Effect of actinomycin D on fisetin-induced facilitation of LTP. The hippocampal slices were untreated (n= 10) or exposed to 1 μM fisetin alone (n= 7) or after a 10-min pretreatment with 40 μM actinomycin D (ACTD, n= 6) and weak tetanic stimulation was applied at time 0. The presentation of the data are as in B and C. ACTD does not block fisetin-dependent facilitation of LTP. [None: 104.6 ± 4.3%; fisetin: 141.9 ± 8.6%, ∗∗, P < 0.01 vs. none; fisetin + ACTD 139.7 ± 13.6%, not significant vs. fisetin (ANOVA followed by Tukey–Kramer test).] All data are the mean ± SEM.

Fig. 5.

Fisetin enhances long-term memory in mice, and the effect of different oral doses of fisetin on object recognition over a 10-min test period is shown. Rolipram, injected i.p. at 0.1 mg/kg, served as a positive control. Data represent the mean ± SEM of 10 mice per treatment group. Data were analyzed by one-way ANOVA followed by post hoc comparisons with Fisher's test. ∗, significant difference from vehicle control (P < 0.02). Vehicle, 51.072 ± 4.293; fisetin 5 mg/kg, 60.820 ± 4.521; fisetin 10 mg/kg, 63.628 ± 4.332; fisetin 25 mg/kg, 66.461 ± 2.984; rolipram, 74.132 ± 2.041. Similar results were obtained in two independent, blinded experiments done by PsychoGenics.

Fig. 6.

Fisetin does not increase cAMP levels in hippocampal slices. Hippocampal slices in ACSF were treated with 1 μM fisetin for 5 min (fisetin) or 3 μM rolipram for 30 min (rolipram) and then either immediately frozen or treated with 5 μM forskolin for an additional 15 min (rolipram + forskolin; fisetin + forskolin) before freezing. Additional slices were treated only with 5 μM forskolin for 15 min (forskolin). The levels of cAMP in the slices were measured by using a scintillation proximity assay, and they are presented as pmol/mg protein ± SD. *, significant difference from control (P < 0.05);#, significant difference from forskolin or rolipram alone (P < 0.005).