Neuroprotective effects of LMW and HMW FGF2 against amyloid beta toxicity in primary cultured hippocampal neurons

Abstract

Basic Fibroblast growth factor (FGF2) is important in development and maintenance of central nervous system function. Studies have demonstrated that low molecular weight (LMW) FGF2 is a neuroprotective factor against various insults in vivo and in vitro. In the present study we investigated the neuroprotective effects of high molecular weight (HMW) and LMW FGF2 against amyloid beta-induced neurotoxicity. The results showed that both LMW and HMW FGF2 attenuated the amyloid beta toxicity in the primary cultured hippocampal neurons as measured by WST and LDH release assay. Moreover, the analysis suggested that HMW FGF2 had stronger neuroprotective effect than LMW FGF2. We then demonstrated that LMW and HMW FGF2 activated the ERK and AKT signaling pathways in a similar way. Furthermore, using the ERK inhibitor and AKT inhibitor, we found that the AKT signaling but not ERK signaling pathway was required for the neuroprotective effects of FGF2. Taken together, these results showed the neuroprotective effects of different forms of FGF2 in an AD model and the mechanism underlying the neuroprotection.

Keywords: AKT; Amyloid beta; HMW FGF2; Hippocampal neuron; LMW FGF2.

Fig. 1

Neuroprotective effects of LMW and HMW FGF2 on Aβ_1–42 toxicity in the primary cultured hippocampal neurons. (A) WST assay showing that the reduced cell viability induced by Aβ_1–42 was inhibited by LMW or HMW FGF2 in a dose dependent manner in the primary cultured hippocampal neurons. One way ANOVA followed by Tukey post-hoc multiple comparisons tests, F=26.58, p<0.05. * compared to the control group, # compared to Aβ_1–42 group. (B) LDH release assay showing that LMW or HMW FGF2 reduced the cytotoxicity induced by Aβ_1–42 in the neurons. One way ANOVA followed by Tukey post-hoc multiple comparisons tests, F=10.4, n=5 p<0.05. * compared to the control group, # compared to Aβ_1–42 group. (C, D) Two way ANOVA analysis showing that HMW FGF2 had stronger neuroprotection than LMW FGF2 in the primary cultured hippocampal neurons. For WST assay: F=30.90, n=5, p<0.05; for LDH release assay: F=10.4, n=5, p<0.05. Three independent experiments were done, data shown represent one experiment.

Fig. 2

FGF receptor is required for the neuroprotection of FGF2. LDH release assay showing that the neuroprotective effects of HMW and LMWFGF2 against Aβ_1–42 were abolished by the FGF receptor 1 inhibitor (SU5402) in the neurons, indicating that FGF receptor activation was required for the neuroprotective effects of FGF2. One way ANOVA followed by Tukey post-hoc multiple comparisons tests, F=20.94, n=5, p<0.05. Two independent experiments were done, data shown represent one experiment.

Fig. 3

LMW and HMW FGF2 activate ERK and AKT in the neurons. (A) Representative western blot bands of phosphorylated ERK after 30 min treatment with FGF2 in the hippocampal neurons. Total ERK served as internal control. (B) Quantification of p-ERK normalized to t-ERK. One way ANOVA followed by Tukey post-hoc multiple comparisons tests, F=7.587, n=4, p<0.05. * compared to the control group. (C) Representative western blot bands of phosphorylated AKT after 30 min treatment with FGF2 in the hippocampal neurons. Total AKT served as internal control. (D) Quantification of p-Akt normalized to t-ERK. One way ANOVA followed by Tukey post-hoc multiple comparisons tests, F=11.89, n=4, p<0.05. Data were pooled from two independent experiments.

Fig. 4

Activation of AKT but not ERK is required for the neuroprotection of FGF2. (A) LDH release assay showing that the rescue effect of FGF2 after Aβ_1–42 induced damage in the neurons was not blocked by ERK inhibitor (U0126), suggesting that ERK activation was not involved in the neuroprotection of FGF2. One way ANOVA followed by Tukey post-hoc multiple comparisons tests, F=19.83, n=4, p<0.05 (B) LDH release assay showing that the rescue effect of FGF2 after Aβ_1–42 induced damage in the neurons was blocked by AKT inhibitor (Ly294002), suggesting that AKT activation was involved in the neuroprotection of FGF2. One way ANOVA followed by Tukey post-hoc multiple comparisons tests, F=21.71, n=5, p<0.05. Two independent experiments were done, data shown represent one experiment.