Lentiviral Modulation of Wnt/β-Catenin Signaling Affects In Vivo LTP

Abstract

Wnt signaling is involved in hippocampal development and synaptogenesis. Numerous recent studies have been focused on the role of Wnt ligands in the regulation of synaptic plasticity. Inhibitors and activators of canonical Wnt signaling were demonstrated to decrease or increase, respectively, in vitro long-term potentiation (LTP) maintenance in hippocampal slices (Chen et al. in J Biol Chem 281:11910-11916, 2006; Vargas et al. in J Neurosci 34:2191-2202, 2014, Vargas et al. in Exp Neurol 264:14-25, 2015). Using lentiviral approach to down- and up-regulate the canonical Wnt signaling, we explored whether Wnt/β-catenin signaling is critical for the in vivo LTP. Chronic suppression of Wnt signaling induced an impairment of in vivo LTP expression 14 days after lentiviral suspension injection, while overexpression of Wnt3 was associated with a transient enhancement of in vivo LTP magnitude. Both effects were related to the early phase LTP and did not affect LTP maintenance. A loss-of-function study demonstrated decreased initial paired pulse facilitation ratio, β-catenin, and phGSK-3β levels. A gain-of-function study revealed not only an increase in PSD-95, β-catenin, and Cyclin D1 protein levels, but also a reduced phGSK-3β level and enhanced GSK-3β kinase activity. These results suggest a presynaptic dysfunction predominantly underlying LTP impairment while postsynaptic modifications are primarily involved in transient LTP amplification. This study is the first demonstration of the involvement of Wnt/β-catenin signaling in synaptic plasticity regulation in an in vivo LTP model.

Keywords: GSK-3β; Hippocampus; LTP; Lentivirus; PSD-95; Paired pulse facilitation; Synaptic plasticity; Wnt signaling.

Fig. 1

The distribution of lentiviral constructs in the hippocampal CA1 area. a The hippocampal formation scheme reprinted from Paxinos and Watson (2005), with permission from Elsevier © 2005. Black and gray rectangles indicate areas, shown on figures b and c, respectively. b, c Epifluorescent images of the hippocampal formations after fluorescent immunostaining with anti-GFP antibodies from rats administered with lentiviral construct expressing dominant-negative Wnt1 (LV-dnWnt1, b) and Wnt3 (LV-Wnt3, c). d, e Wnt1 (d) and Wnt3 (e) expression levels in the hippocampus 14 days after lentiviral injections of LV-dnWnt1 (d, n = 13) and LV-Wnt3 (e, n = 8). Each bar represents the mean ± SEM percentage from LV-GFP group. ^# p < 0.01 (Mann–Whitney U test)

Fig. 2

Long-term potentiation (LTP) in vivo in the Schaffer collateral-CA1 synapses 14 days after lentiviral injections. a, c The time course of normalized fEPSP amplitudes in LV-dnWnt1 (a), LV-Wnt3 (c) and LV-GFP groups for 60 min before (baseline) and 180 min after high-frequency stimulation (HFS). Each point represents the mean ± SEM percentage from baseline; arrows denote the HFS applied for inducing LTP. *p < 0.05; ^# p < 0.01 in LV-dnWnt1 (n = 9) and LV-Wnt3 (n = 9) versus LV-GFP (n = 10) group (mixed design ANOVA followed by Fisher’s LSD post hoc test). b, d Long-term potentiation in the LV-dnWnt1 group (b) and LV-Wnt3 group (d) versus LV-GFP group at 20, 60, and 180 min after HFS. Each bar represents the mean ± SEM percentage from baseline. ^# p < 0.01 in LV-dnWnt1 (n = 9) and LV-Wnt3 (n = 9) versus LV-GFP (n = 10) group (Student’s t test). e Individual traces of fEPSP in LV-GFP (left plot), LV-dnWnt1 (middle plot), and LV-Wnt3 (right plot) groups before (dotted lines) and after (filled lines) HFS

Fig. 3

Paired pulse facilitation (PPF) ratio in the Schaffer collateral-CA1 synapses 14 days after lentiviral injections. a The time course of normalized PPF ratio for 60 min before (baseline) and 180 min after HFS. Each point represents the mean ± SEM. *p < 0.05 in LV-dnWnt1 versus LV-GFP group, numbers above some points represent p value (mixed design ANOVA followed by Fisher’s LSD post hoc test). b The average PPF ratio in the baseline (initial PPF ratio) in LV-dnWnt1 (n = 9), LV-Wnt3 (n = 9), and LV-GFP (n = 10) groups. Each bar represents the mean ± SEM. *p < 0.05; ^# p < 0.01 (Mann–Whitney U test). c Analysis of relations between changes in initial PPF ratios and LTP magnitudes (10–40 min after HFS). The linear regression lines are given for each group: LV-GFP (n = 10) y = 106.50 + 21.08 * x; LV-dnWnt1 (n = 9) y = 113.26 − 0.55 * x; and LV-Wnt3 (n = 9) y = 70.33 + 51.07 * x. Coefficients of correlations (r) are given on the plot for each group

Fig. 4

PSD-95 expression level in the hippocampus 14 days after lentiviral injections of LV-dnWnt1 (a, n = 9) and LV-Wnt3 (b, n = 9). Each bar represents the mean ± SEM. *p < 0.05 (Mann–Whitney U test)

Fig. 5

β-catenin (a, b), Cyclin D1 (c, d), and c-Myc (e, f) expression levels in the hippocampus 14 days after lentiviral injections of LV-dnWnt1 (a, n = 9; c, n = 12; e, n = 12) and LV-Wnt3 (b, n = 10; d, n = 14; f, n = 15/group). Each bar represents the mean ± SEM. *p < 0.05 (Mann–Whitney U test)

Fig. 6

a–d GSK-3β (a, b) and phosphorylated at serine 9 GSK-3β (c, d) expression levels in the hippocampus 14 days after lentiviral injections of LV-dnwnt1 (a, c, n = 9–10) and LV-Wnt3 (b, d, n = 13–14). e GSK-3β kinase activity in the hippocampus is increased in both LV-dnwnt1 (n = 6) and LV-Wnt3 (n = 6) versus LV-GFP (n = 6) group. Each bar represents the mean ± SEM. *p < 0.05 (Mann–Whitney U test)