A mouse model for MeCP2 duplication syndrome: MeCP2 overexpression impairs learning and memory and synaptic transmission

Abstract

Rett syndrome and MECP2 duplication syndrome are neurodevelopmental disorders that arise from loss-of-function and gain-of-function alterations in methyl-CpG binding protein 2 (MeCP2) expression, respectively. Although there have been studies examining MeCP2 loss of function in animal models, there is limited information on MeCP2 overexpression in animal models. Here, we characterize a mouse line with MeCP2 overexpression restricted to neurons (Tau-Mecp2). This MeCP2 overexpression line shows motor coordination deficits, heightened anxiety, and impairments in learning and memory that are accompanied by deficits in long-term potentiation and short-term synaptic plasticity. Whole-cell voltage-clamp recordings of cultured hippocampal neurons from Tau-Mecp2 mice reveal augmented frequency of miniature EPSCs with no change in miniature IPSCs, indicating that overexpression of MeCP2 selectively impacts excitatory synapse function. Moreover, we show that alterations in transcriptional repression mechanisms underlie the synaptic phenotypes in hippocampal neurons from the Tau-Mecp2 mice. These results demonstrate that the Tau-Mecp2 mouse line recapitulates many key phenotypes of MECP2 duplication syndrome and support the use of these mice to further study this devastating disorder.

Figure 1.

Increased expression of MeCP2 in Tau–Mecp2 mice compared with WT control littermates. A, B, Immunofluorescence images of the CA1 and dentate gyrus (DG) areas of the hippocampus. Left column shows MeCP2 protein in WT control littermates. Right column shows MeCP2 in Tau–Mecp2 mice. Top row is at a 10× magnification, and the bottom row is at a 40× magnification. C, Western blot data demonstrating an increase in MeCP2 protein expression in Tau–Mecp2 mice in different areas of the brain. FC, Frontal cortex; CPu, caudate–putamen; HC, hippocampus; CBL, cerebellum. WT mice, n = 4; Tau–Mecp2 mice, n = 4. *p < 0.05.

Figure 2.

Behavioral phenotypes observed in mice that overexpress Mecp2. A, Locomotor activity is not significantly different between WT and Tau–Mecp2 mice. B, Tau–Mecp2 mice spend significantly less time on the rotarod on trials 2–4, 6, and 7 compared with WT mice, demonstrating motor coordination deficits. Significant main effects of time (F_(7,315) = 19.5, p < 0.05) and genotype (F_(7,315) = 8.2, p < 0.05). Post hoc Fisher's LSD tests revealed significant differences between Tau–Mecp2 and WT mice at trials 2–4, 6, and 7. C, DL test demonstrating an increased anxiety-like phenotype in mice that overexpress Mecp2 compared with WT mice (t₍₂₁₎ = 4.99, p < 0.05). Tau–Mecp2 mice spend significantly less time in the light side than WT mice. D, EPM data showing increased anxiety-like behavior in Tau–Mecp2 mice, which spend less time in the open arms and more time in the closed arms compared with WT controls (t₍₂₁₎ = 3.30, p < 0.05). WT mice, n = 11;Tau–Mecp2 mice, n = 12. *p < 0.05 between Tau–Mecp2 and WT mice.

Figure 3.

Learning and memory deficits in Tau–Mecp2 mice. A, Tau–Mecp2 mice (n = 12) show increased freezing responses in both context and cue fear conditioning compared with WT (n = 11) littermates (t₍₂₀₎ = 7.4, t₍₂₀₎ = 6.3, p < 0.05, respectively). B, Tau–Mecp2 mice do not learn to extinguish freezing behavior to repeated presentations of cue after nine extinction sessions, whereas WT control littermates extinguish freezing responses by the fourth extinction session, indicating learning and memory deficits in Tau–Mecp2 mice (WT, n = 7; Tau–Mecp2, n = 6). A repeated-measures ANOVA revealed a significant interaction effect between trials and genotype (F_(8,125) = 4.8, p < 0.05), and a Fisher's LSD analysis revealed significant differences between all extinction trials. C, Mice that overexpress Mecp2 are not able to distinguish between a novel object and a familiar one. Tau–Mecp2 mice spend less time exploring the novel object than WT mice (t₍₂₂₎ = 3.4, p < 0.05) and also spend less time with the novel object versus the familiar object as demonstrated by their significant negative difference score (DS) (t₍₂₂₎ = 2.1, p < 0.05). There is no difference, however, in the amount of time spent with the familiar object between Tau–Mecp2 and WT mice. *p < 0.05 between WT mice and Tau–Mecp2 mice.

Figure 4.

LTP induced by HFS is impaired in Tau–Mecp2 hippocampal slices. A, Tau–Mecp2 mice show attenuated LTP responses induced by HFS compared with WT controls (WT, n = 16; Tau–Mecp2, n = 11). A significant time × genotype interaction effect was seen (F_(271,7343) = 3.0, p < 0.05). Post hoc comparisons using Fisher's LSD revealed differences at almost all time points after HFS except for 15 time points of the 78 time points analyzed. Representative traces are shown in the inset (black, before stimulation; red, after LTP stimulation). B, There were no significant differences in the slope of the I/O relationship between Tau–Mecp2 and WT mice (WT, n = 8; Tau–Mecp2, n = 10). C, Tau–Mecp2 hippocampal slices show impairments in PPF. PPF was significantly augmented by MeCP2 overexpression, with Tau–Mecp2 mice showing increased PPF at interstimulus intervals 30 ms (t₍₁₆₎ = 2.2, p < 0.05) and 50 ms (t₍₁₆₎ = 3.1, p < 0.05) compared with WT controls, indicating a decreased probability of neurotransmitter release (WT, n = 8; Tau–Mecp2, n = 10). *p < 0.05 between WT mice and Tau–Mecp2 mice.

Figure 5.

Increased excitatory neurotransmission in mice that overexpress Mecp2. A, Representative traces of mEPSC frequency in WT and Tau–Mecp2 hippocampal neuronal cultures. B, mEPSC frequency was significantly increased in hippocampal Tau–Mecp2 cultures compared with WT cultures (*p < 0.05). C, There were no significant differences in the cumulative distribution of mEPSC amplitudes. D, Representative traces of mIPSC frequency in WT and Tau–Mecp2 hippocampal neurons. E, There were no significant differences in mIPSC frequency. F, Cumulative distribution of mIPSC amplitudes was not significantly altered in Tau–Mecp2 hippocampal neurons. The number of cells recorded in mEPSC and mIPSC experiments are shown in the respective bar graph.

Figure 6.

Inhibition of transcriptional repressor processes rescue the synaptic deficits in hippocampal neurons from Tau–Mecp2 mice. A, BDNF protein expression was quantified relative to actin in Tau–Mecp2 and WT mice. There were no significant differences in relative BDNF protein expression in Tau–Mecp2 compared with WT mice (n = 4 for each group). B, Left, Representative traces of mEPSC frequency in WT and Tau–Mecp2 hippocampal neuronal cultures treated with TSA. B, The significant increase in mEPSC frequency seen in Tau–Mecp2 hippocampal neurons is reduced back to WT levels with 24 h TSA treatment (WT, n = 9; Tau–Mecp2, n = 8; Tau + TSA, n = 8; *p < 0.05).