Regulation of neural differentiation, synaptic scaling and animal behavior by MeCP2 phophorylation

Abstract

Highly expressed in the mammalian brain and widely distributed across the genome, MeCP2 is a key player in recognizing modified DNA and interpreting the epigenetic information encoded in different DNA methylation/hydroxymethylation patterns. Alterations in sequence or copy number of the X-linked human MECP2 gene cause either Rett syndrome (RTT) or MECP2 duplication syndrome. Alterations in MECP2 levels have also been identified in patients with autism. To fully understand the significant role of MECP2 in regulating the development and function of the nervous system, it is important to study all aspects of MeCP2 function. Stimulus-induced MeCP2 phosphorylation has been shown to influence the proliferation and differentiation of neural progenitor cells, synaptic scaling, excitatory synaptogenesis, and animal behavior. However, all of the previous functional evidence is from studying phospho-dead mutations. In addition, the relationship between phosphorylation events at multiple sites on the MeCP2 protein is not well understood. Here, we report the generation of a phospho-mimic knockin Mecp2 mouse line. At the synaptic and behavioral levels, the phospho-mimic Mecp2 mice show phenotypes opposite to those observed in phospho-dead mutation at the same phosphorylation site. Moreover, we report opposite phenotypes between phospho-mutants of two sites on the MeCP2 protein. Our new data further confirm the functional significance of specific MeCP2 phosphorylation event and support the opposing regulatory role between different MeCP2 phosphorylation events.

Keywords: MeCP2; Phosphorylation; Rett syndrome; S421; S80.

Figure 1. Generation and characterization of the Mecp2^S421E/y mice

(a) Genomic DNA from wild type (+/y) and Mecp2^S421E/y mice (S421E/y) was amplified by PCR and sequenced, showing the wild type sequence TCA (encoding serine) is mutated to GAG (encoding glutamie acid) in the Mecp2^S421E/y mice. (b) Western blot analysis with antibodies specific for phospho-S421 (pS421) and total MeCP2 in brain nuclear fraction from Mecp2^+/y, Mecp2^{S421A;S424A/y}, and Mecp2^S421E/y mice. A nuclear protein TDP43 was used as a sample loading control. (c) Quantification of the total MeCP2 protein level in wild type and Mecp2^S421E/y cortex (n=3 mice in each genotype, p=0.18). (d) The Mecp2^S421E/y mice show a deficit in the hippocampus-dependent contextual fear learning/memory (n=4 mice in wild type and n=5 mice in Mecp2^S421E/y, p=0.05). *p<0.05, Bar graphs represent mean ± s.e.m.

Figure 2. Reduced synaptogenesis in the Mecp2^S421E/y mice

(a) Representative images of wild type (WT) and Mecp2^S421E/y (S421E) primary hippocampal neurons labeled with MAP2, VGLUT1, and PSD95. (b) Representative images of the primary dendrites of WT and S421E primary hippocampal neurons labeled with MAP2, VGLUT1, and PSD95. (c) Quantification of the density of excitatory synapses as measured by the colocalization of pre- and post-synaptic densities in hippocampal neurons isolated from the WT and S421E mice (n=52 in each genotype, p=0.729 for VGLUT1, p=0.195 for PSD95, p=0.007 for colocalization of VGLUT1 and PSD95). **p<0.01, Bar graphs represent mean ± s.e.m.

Figure 3. Impaired TTX-induced synaptic scaling up and normal bicucullin-induced synaptic scaling down in hippocampal neurons isolated from the Mecp2^S421E/y mice

(a–c) Representative traces (a), average waveforms (b) and cumulative probability distribution (c) of miniature excitatory postsynaptic current (mEPSC) obtained through whole-cell patch clamp recording from hippocampal neurons isolated from wild type (WT) and Mecp2^S421E/y (S421E) mice that were cultured for 18 days in vitro (18 DIV) and treated with either control solution (control) or 1 μm TTX (TTX) for 48 hours. (d–e) Quantification of mean mEPSC amplitute (d) and frequency (e) for each experimental group in (a–c). (n=74–80 cells per group). (f–h) Representative traces (f), average waveforms (g) and cumulative probability distribution (h) of miniature excitatory postsynaptic current (mEPSC) obtained through whole-cell patch clamp recording from hippocampal neurons isolated from wild type (WT) and Mecp2^S421E/y (S421E) mice that were cultured for 18 days in vitro (18 DIV) and treated with either control solution (control) or 40 μm bicuculline (bicuculline) for 48 hours. (i–j) Quantification of mean mEPSC amplitute (i) and frequency (j) for each experimental group in (f–h). (n=52–58 cells per group). *p<0.05, **p<0.01. Bar graphs represent mean ± s.e.m.

Figure 4. Impaired spatial learning and memory in the Mecp2^S80A/y mice

(a) Escape latency during the training sessions in the hidden-platform version of Morris water maze. The Mecp2^S80A/y mice (S80A/y) did not learn as well as the wild type mice (+/y). (b) Percentage of time spent in each quadrant of the water maze during probe trial. While the +/y mice remembered the location of the hidden platform as indicated by their spending significantly more time in the target quadrant to search for the platform, the S80A/y mice did not. (c) The number of times mice crossing the location of the hidden platform used to be during the training sessions is significantly smaller in the S80A/y mice. (d) The S80A/y mice learned the location of the platform as efficiently as the +/y mice in the visible-platform version of the water maze, suggesting the learning dificit cannot be attributed to vision deficit. 8 Mecp2^S80A/y mice and 7 wild type littermates were included in the Morris water maze test. *p<0.05, **p<0.01. Bar graphs represent mean ± s.e.m.

Figure 5. Increased proliferation and decreased neural differentiation in the Mecp2^S80A/y aNPCs

(a) Representative images and quantification of BrdU positive aNPCs isolated from wild type (+/y) and Mecp2^S80A/y (S80A/y) adult hippocampus under proliferation condition with BrdU pulse labeling, followed by immunocytochemistry analysis using antibody specific for BrdU (p=0.04, n=3 in each genotype). BrdU labeling indicates actively dividing cells. (b) Representative immunocytochemistry images and percentage quantification of Tuj1 positive neurons differentiated from wild type (+/y) and Mecp2^S80A/y (S80A/y) aNPCs (p=0.002, n=4 in each genotype). Tuj1 is a cell type specific marker for neurons. (c) Representative immunocytochemistry images and percentage quantification of GFAP positive astrocytes differentiated from wild type (+/y) and Mecp2^S80A/y (S80A/y) aNPCs (p=0.68, n=3 in each genotype). GFAP is a cell type specific marker for astrocytes. Scale bars = 50 μm. *p<0.05, Bar graphs represent mean ± s.e.m.