Suppression of neuronal AMPKβ2 isoform impairs recognition memory and synaptic plasticity

Abstract

AMP-activated protein kinase (AMPK) is an αβγ heterotrimer protein kinase that functions as a molecular sensor to maintain energy homeostasis. Accumulating evidence suggests a role of AMPK signaling in the regulation of synaptic plasticity and cognitive function; however, isoform-specific roles of AMPK in the central nervous system (CNS) remain elusive. Regulation of the AMPK activities has focused on the manipulation of the α or γ subunit. Meanwhile, accumulating evidence indicates that the β subunit is critical for sensing nutrients such as fatty acids and glycogen to control AMPK activity. Here, we generated transgenic mice with conditional suppression of either AMPKβ1 or β2 in neurons and characterized potential isoform-specific roles of AMPKβ in cognitive function and underlying mechanisms. We found that AMPKβ2 (but not β1) suppression resulted in impaired recognition memory, reduced hippocampal synaptic plasticity, and altered structure of hippocampal postsynaptic densities and dendritic spines. Our study implicates a role for the AMPKβ2 isoform in the regulation of synaptic and cognitive function.

Keywords: AMPK; Isoform; LTP; Memory; Mouse model; Synaptic plasticity.

Fig. 1.. AMPKβ2 suppression impairs recognition memory.

(A-B) Representative PCR (A) and hippocampal western blot (B) of the generation of a heterozygous (+/−) or homozygous (−/−) mouse model of AMPKβ1 or AMPKβ2 suppression. (C) Cre and β1^+/− mice appear to spend a greater time with the NO, while β2^+/− mice appear to spend equal time with both objects. (D) β2^+/− mice display an impaired ability to discriminate between NO and FO, as assessed by the discrimination index (DI (t_N + t_F) ÷ t_T). n = 14–17 per group; *p < 0.05. One-way ANOVA with Tukey’s post-hoc test, F_2,43 = 4.658. (E-F) Unchanged OF performance between groups. n = 11–12 per group. (G) Unaltered MWM five-day learning curve between groups. n = 11–12 per group. (H—I) Probe trial yielded no change in time spent in target quadrant or “platform” crossings. n = 11–12 per group. (J) Mice in all groups display normal VP performance. n = 11–12 per group.

Fig. 2.. Suppression of AMPKβ2 impairs hippocampal LTP and alters AMPKα phosphorylation.

(A) Hippocampal high-frequency stimulation (HFS)-induced long-term potentiation (LTP) in Cre mice is conserved in β1^+/− mice but dampened in β2^+/− mice. N = 3, n = 6–8 slices per group. (B) β2^+/− mice display significantly reduced fEPSP at 90 min. Post-HFS. N = 3 mice, n = 6–8 slices per group; *p = 0.0106, **p = 0.0039. One-way ANOVA with Tukey’s post-hoc test, F_2,19 = 8.486. (C) Unaltered paired pulse facilitation (PPF) between all groups. N = 3 mice, n = 6–8 slices per group. (D) Unchanged input-output response between groups. N = 3 mice, n = 6–8 slices per group. (E) Western blot indicates impaired AMPKα phosphorylation at Thr¹⁷². n = 8–11 per group; *p = 0.0137. One-way ANOVA with Tukey’s post-hoc analysis, F_2,25 = 4.743. (F) Mice in all groups display similar protein translation as assessed by the SUnSET assay. n = 6 per group. Vertical line indicates a break in the gel for the representative image.

Fig. 3.. Altered postsynaptic morphology of CA1 dendrites in β2^+/− mice.

(A) Representative TEM images. Arrows indicate PSDs. Scale = 500 nm. (B) Number of PSDs counted per 10 μm². N = 3 mice, n = 9 grid sections per group; *p = 0.0125. One-way ANOVA with Tukey’s post-hoc analysis, F_2,24 = 4.890. (C) Quantification of PSD area. N = 3 mice, n = 15 images per group; **p = 0.0048, ***p = 0.0005. One-way ANOVA with Tukey’s post-hoc analysis, F_2,42 = 9.600. (D) Classification of immature (1, filopodia; 2, long-thin; 3, thin) and mature (4, mushroom; 5, stubby; 6, branched) spines and representative Golgi-Cox images. Scale = 10 μm. (E) Total density of spines per 10 μm. N = 3 mice, n = 45 images per group; *p = 0.0382, **p = 0.0071. One-way ANOVA with Tukey’s post-hoc test, F_2,132 = 5.321. (F-G) Density of mature and immature spines. N = 3 mice, n = 45 images per group; *p = 0.397, **p = 0.0091, ****p < 0.0001. One-way ANOVA with Tukey’s post-hoc analysis, F_2,132 = 27.39 (F), F_2,132 = 13.46 (G). (H) Quantification of spine maturity as expressed by mature-to-immature ratio. N = 3 mice, n = 45 images per group; ****p < 0.0001. One-way ANOVA with Tukey’s post-hoc analysis, F_2,132 = 32.32.

Fig. 4.. Homozygous knockout mice display similar recognition memory and hippocampal LTP impairments to heterozygous knockdown mice.

(A) Cre and β1^−/− mice appear to spend more time with the NO than the FO. (B) β2^−/− mice display an impairment in their ability to discriminate between NO and FO, as assessed by the discrimination index. n = 14—16 per group; *p < 0.05. One-way ANOVA with Tukey’s post-hoc test, F_2,41 = 4.530. (C—D) Unchanged OF performance between groups. n = 11–12 per group. (E-G) Unaltered MWM learning curve, time spent in target quadrant, or “platform” crossings. n = 11–12 per group. (H) Mice in all groups display normal VP performance. n = 11 per group. (I) Hippocampal high-frequency stimulation (HFS)-induced long-term potentiation (LTP) is dampened in β2^−/− mice, not β1^−/− when compared to Cre. N = 3 mice, n = 8 slices per group. (J) β2^−/− mice display significantly reduced fEPSP at 90 min. Post-HFS. N = 3 mice, n = 8 slices per group; *p = 0.0333, **p = 0.0048. One-way ANOVA with Tukey’s post-hoc test, F_2,21 = 7.364. (K) Western blot of p-AMPKα (Thr¹⁷²) in homozygous knockout mice. n = 8 per group.