MeCP2 Is Critical for Maintaining Mature Neuronal Networks and Global Brain Anatomy during Late Stages of Postnatal Brain Development and in the Mature Adult Brain

The loss of MeCP2 at late juvenile or adult stages induces RTT-like phenotypes in male and female mice. A, B, Phenotypic scores and Kaplan–Meier survival time course, respectively, of 5W-Tam-injected Mecp2^loxJ/y/CreER and the indicated control mice. The number of mice in B is similar to the indicated in A. D, E, Phenotypic scores and Kaplan–Meier survival time course, respectively, of 5W-Tam-injected Mecp2^loxB/y/CreER and the indicated control mice. The number of mice in E is similar to the indicated in D. G, H, Phenotypic scores and weight gain time course, respectively, of 5W-Tam-injected Mecp2^loxJ/+/CreER female mice and the indicated control mice. The number of mice in H is similar to G. J, K, Phenotypic scores and Kaplan–Meier survival time course graphs, respectively, of 10W-Tam-injected Mecp2^loxJ/y/CreER and the indicated control mice. The number of mice in K is similar to the indicated in J. M, Overlay of the aggregate symptom scores from the time of injection of the 5W-Tam-injected Mecp2^loxJ/y/CreER and the 10W-Tam-injected Mecp2^loxJ/y/CreER mice. Inset shows no significant differences in the mean of the trend slopes between the 5W- and 10W-Tam-injected mice (NS, p > 0.05). Error bars are mean ± SEM. t test was used to compare within the 5W- and the 10W-Tam-injected groups. N, Overlay of the survival curves of the 5W- and 10W-Tam-injected Mecp2^loxJ/y/CreER mice. A LogRank survival test showed no significant difference between the survival time of the 5W- and the 10W-Tam-injected Mecp2^loxJ/y/CreER mice. Inset shows no significant differences in the mean age of death between the 5W- and 10W-Tam-injected mice (NS, p > 0.05). Error bars are mean ± SEM. t test was used to compare within the 5W- and the 10W-Tam-injected groups. C, F, I, L, Images illustrating the hindlimb clasping phenotype of the indicated Tam-injected MeCP2^loxJ/CreER or Tam-injected MeCP2^loxB/CreER mice. Note that the Tam-injected MeCP2^loxJ/y/CreER mice (male and female) are overweight and that the female is injured due to overgrooming (white arrow). All the mice in the images were injected with Tam at 5 weeks of age except in L, where the mouse was injected at 10 weeks of age (10 W). Red arrows show the time of Tam or Veh injection.

Mice that lost MeCP2 at a late juvenile or adult stage show similar behavioral deficits. Male and female mice were subjected for the indicated behavioral tests at 13 weeks or 27 weeks after injection, respectively. A–K, Total activity test in digiscan activity monitor (A–C), wire-hang test (D–F), rotarod test (G–I), and dowel test (J, K). The colors of the graphs and bar graphs correspond to the indicated genotypes in the right bottom corner. A, D, G, J, 5W-Tam-injected Mecp2^loxJ/y/CreER and the indicated control mice. B, E, H, K, 5W-Tam-injected Mecp2^loxJ/+/CreER and the indicated control female mice. C, F, I, 10W-Tam-injected Mecp2^loxJ/y/CreER and the indicated control male mice. The number of mice in D and G is similar to that indicated in A; in E and H it is similar to B; in F and I it is similar to C. Note that one outlier 10W-Tam-injected Mecp2^loxJ/y/CreER mouse whose score was more than two SDs from the group mean was removed from the rotarod analysis in I. One-way ANOVA followed by appropriate post hoc for multiple-comparisons test was used to determine differences between groups. Error bars are mean ± SEM. *p < 0.05 represents significant differences between the different Tam-injected Mecp2^loxJ/CreER mouse groups and all control littermate groups.

The loss of MeCP2 at late juvenile or adult stage leads to shrinkage of the brain. A, B, Representative images showing that symptomatic Mecp2^loxJ/y/CreER mice (loxJ/y/Cre+T), injected with Tam at 5 (A) or 10 (B) weeks of age, have smaller brains than their control littermates, Mecp2^loxJ/y+T (loxJ/y+T) and Mecp2^loxJ/y/CreER+V (loxJ/y/Cre+V) mice. Brains are from mice at 16 weeks after injection. C, D, Age-dependent changes in brain weights of WT, 5W- (C) or 10W- (D) Tam- or Veh-injected Mecp2^loxJ/y/CreER and Mecp2^loxJ/y mice. Fragmented lines distinguish between the brains that are smaller (below) or bigger (above) than the Mecp2^loxJ/y/CreER brains right after the last Tam injection (6W or 11W). E, Brains of the 5W-Tam-injected Mecp2^loxJ/y/CreER mice are smaller than their control littermates at 24 weeks of age (18 weeks after injection), and smaller than the WT brains at 5 weeks of age and the 5W-Tam-injected Mecp2^loxJ/y/CreER at 6 weeks of age (after the last Tam injection). F, Brains of the 10W-Tam-injected Mecp2^loxJ/y/CreER mice are smaller than the brains of the 10W-Veh-injected Mecp2^loxJ/y/CreER mice at 29–32 weeks of age (18–21 weeks after injection) and smaller than the WT brains at 10 weeks of age and the 10W-Tam-injected Mecp2^loxJ/y/CreER brains at 11 weeks of age (after the last Tam injection). G, Brains of the 5W-Tam-injected Mecp2^loxJ/+/CreER female mice are smaller than the brains of their control littermates at 39–50 and 79–99 weeks of age. Two-way ANOVA analysis followed by appropriate post hoc for multiple-comparison tests were performed to determine differences between Veh/Tam groups and between the different age groups. Error bars are mean ± SEM. *p < 0.05. n = 4–6 mice per genotype. H, I, Time course analysis of body weight gain of 5W- (H) or 10W- (I) Tam-injected Mecp2^loxJ/y/CreER mice and the indicated control male mice. n indicates the number of mice used. Red arrows indicate the time of Tam or Veh injection.

The brain anatomy is severely affected upon the loss of MeCP2 at a late juvenile or adult stage. A, B, Images of Nissl staining showing smaller brain structures for the 5W- (A) and 10W- (B) Tam-injected mice at 24 and 30 weeks of age, respectively, when compared with their control Veh-injected littermates. CTX, Cortex; LV, lateral ventricle; HP, hippocampus; TH, thalamus; HY, hypothalamus. Scale bars, 800 μm. C, D, Representative images of Nissl staining showing smaller hippocampal structure for the 5W- (C) and 10W- (D) Tam-injected mice. DG, Dentate gyrus. Scale bars, 400 μm. E, F, The cell bodies of the pyramidal (Py) neurons in the CA1 area of the hippocampus of the 5W- (E) and 10W- (F) Tam-injected Mecp2^loxJ/y/CreER mice are more compact. so, Stratum oriens; py, pyramidal layer; sr, radiatum. Scale bars, 20 μm. G, H, The thickness of the pyramidal neuronal cell bodies in the CA1 area of the 5W- (G) and 10W- (H) Tam-injected Mecp2^loxJ/y/CreER mice is significantly reduced. I, J, Representative images of Nissl staining showing a higher density of neuronal cell bodies in layer V of the motor cortex of the 5W- (I) or 10W- (J) Tam-injected mice when compared with 5W- or 10W-Veh-injected Mecp2^loxJ/y/CreER mice. Scale bars, 40 μm. K, L, The density of the neurons in layer V of the motor cortex of 5W-(K) and the 10W- (L) Tam-injected Mecp2^loxJ/y/CreER mice is significantly higher than the Veh-injected control mice. t test was used to compare within Veh and Tam groups. Error bars are mean ± SEM. *p < 0.05. n = 3 mice per genotype.

Dendritic complexity of hippocampal pyramidal neurons is severely affected upon the loss of MeCP2 at late juvenile stage. A, Representative Camera lucida tracing of CA1 pyramidal neurons in the hippocampus of symptomatic 5W-Tam-injected Mecp2^loxJ/y/CreER and the 5W-Veh-injected Mecp2^loxJ/y/CreER control littermate mice. Concentric circles for Sholl analysis in 10 μm radius increments are superimposed in red. Scale bars, 40 μm. B, E, Sholl analysis of the 5W-Tam-injected mice showing that the basal (B) and apical (E) dendrites of CA1 pyramidal neurons at 18 weeks after the last Tam injection (24 weeks of age) are significantly less complex than those of the 5W-Veh-injected Mecp2^loxJ/y/CreER age-matched littermate mice and even less complex than the dendrites of Tam-injected mice right after the last injection (6 weeks of age). Two-way repeated-measures ANOVA followed by appropriate post hoc for multiple-comparisons test was used to determine differences within Veh and Tam groups at 18 weeks after injection or within 0 and 18 weeks after the last Tam injection of 5W-Tam-injected Mecp2^loxJ/y/CreER mice. C, D, F, G, CA1 pyramidal neurons of 5W-Tam-injected Mecp2^loxJ/y/CreER mice exhibit fewer and shorter basal (C, D) and apical (F, G) dendritic branches at 18 weeks after Tam injection. Mann and Whitney test was used to compare within Veh and Tam groups at 18 weeks after injection or within the 5W-Tam-injected Mecp2^loxJ/y/CreER mice at 0 and 18 weeks after injection. H, Representative images of Camera lucida tracing of CA1 pyramidal neurons in the hippocampus showing structural abnormalities in spines and the associated secondary dendrites of the 5W-Tam-injected Mecp2^loxJ/y/CreER mice when compared with Veh-injected Mecp2^loxJ/y/CreER or Tam-injected wild-type (WT) mice, 18 weeks after injection. Arrows indicate swelling or thickening of the associated dendritic branches. Scale bars, 2 μm. I, CA1 pyramidal neurons of 5W-Tam-injected Mecp2^loxJ/y/CreER mice demonstrate significant reduction in dendritic spine density. Spine density per 100 μm length was measured on secondary dendrites. One-way ANOVA followed by appropriate post hoc for multiple-comparison tests was used to determine differences between groups. Error bars are mean ± SEM. *p < 0.05 significant differences in dendritic complexity of the 5W-Tam-injected Mecp2^loxJ/y/CreER at 0 and 18 weeks after the last injection. ^#p < 0.05 significant differences between 5W-Tam and Veh-injected Mecp2^loxJ/y/CreER mice at 24 weeks of age (18 weeks after injection). n = 3 mice per genotype and age. n = 5–6 neurons per animal.

The loss of MeCP2 at adult stage induces reduction in dendritic complexity and spine density. A, D, Sholl analysis of the 10W-Tam-injected mice showing that the basal (A) and apical (D) dendrites of CA1 pyramidal neurons at 29 weeks of age (18 weeks after Tam injection) are significantly less complex than those of 11 weeks of age (right after the last Tam injection) and less complex than the dendrites of the 10W-Veh-injected Mecp2^loxJ/y/CreER age-matched littermates. Two-way repeated-measures ANOVA followed by appropriate post hoc for multiple-comparisons test was used to determine differences within Veh and Tam groups at 18 weeks after injection or within 0 and 18 weeks after the last Tam injection of 10W-Tam-injected Mecp2^loxJ/y/CreER mice. B,C, E, F, The CA1 pyramidal neurons of 10W-Tam-injected Mecp2^loxJ/y/CreER mice exhibit fewer and shorter basal (B, C) and apical (E, F) dendritic branches at 29 weeks of age (18 weeks after injection). Mann and Whitney test was used to compare within Veh and Tam groups at 18 weeks after injection or within the 10W-Tam-injected Mecp2^loxJ/y/CreER mice at 0 and 18 weeks after injection. G, Representative images of Camera lucida tracing of CA1 pyramidal neurons in the hippocampus showing structural abnormalities in spines and the associated secondary dendrites of the 10W-Tam-injected Mecp2^loxJ/y CreER mice when compared with Veh-injected Mecp2^loxJ/y/CreER mice, 18 weeks after injection. Arrows indicate swelling or thickening of the associated dendritic branches. Scale bars, 2 μm. H, CA1 pyramidal neurons of 10W-Tam-injected Mecp2^loxJ/y/CreER mice demonstrate significant reduction in dendritic spine density. Spine density per 100 μm length was measured on secondary dendrites. t test was used to compare within Veh and Tam groups. Error bars are mean ± SEM. *p < 0.05 significant differences in dendritic complexity of the 10W-Tam-injected Mecp2^loxJ/y/CreER at 0 and 18 weeks after the last injection. ^#p < 0.05 significant differences between 10W-Tam and Veh-injected Mecp2^loxJ/y/CreER mice at 29 weeks of age (18 weeks after injection). n = 3 mice per genotype and age. n = 5–6 neurons per animal.

Processes projected from hippocampal astrocyte cell bodies are severely affected upon the loss of MeCP2 at late juvenile stage. A, Representative Camera lucida tracing of astrocytes in CA1 area of the hippocampus of symptomatic 5W-Tam-injected Mecp2^loxJ/y/CreER and the 5W-Veh-injected Mecp2^loxJ/y/CreER control littermate mice. Concentric circles for Sholl analysis in 10 μm radius increments are superimposed in red. Scale bars, 20 μm. B, Sholl analysis of the 5W-Tam-injected mice showing that the processes of hippocampal astrocytes at 18 weeks after the last Tam injection (24 weeks of age) are significantly less complex than those of the 5W-Veh-injected Mecp2^loxJ/y/CreER age-matched littermate mice. Two-way repeated-measures ANOVA followed by appropriate post hoc for multiple-comparisons test was used to determine differences within Veh and Tam groups at 18 weeks after injection. C, D, Hippocampal astrocytes of symptomatic 5W-Tam-injected Mecp2^loxJ/y/CreER mice exhibit fewer (C) and shorter (D) branches at 18 weeks after Tam injection. t test was used to compare between Veh and Tam groups. ^#p < 0.05 significant differences between 5W-Tam and Veh-injected Mecp2^loxJ/y/CreER mice at 24 weeks of age (18 weeks after injection). Error bars are mean ± SEM. n = 3 mice per genotype. n = 5 astrocytes per animal.

The loss of MeCP2 at late juvenile and adult stage results in specific reduction of synaptic protein expression in symptomatic Tam-injected Mecp2^loxJ/y/CreER mice. A, Western blot showing significant reduction in the levels of MeCP2, and the following synaptic proteins: CaMKIIα, CaMKIIβ, GluR2/3, GABABR2, Vglut1, NMDAR2A, and Synapsin 1 in the whole brain of symptomatic 5W-Tam-injected Mecp2^loxJ/y/CreER mice when compared with their levels in the brains of the 5W-Tam-injected Mecp2^loxJ/y or the 5W-Veh-injected Mecp2^loxJ/y/CreER mice. B–D, Western blot showing significant reduction in the levels of CaMKIIα protein in the indicated areas of the brain of symptomatic Tam-injected Mecp2^loxJ/y/CreER (3) male mice injected at 5 (B) or 10 (C) weeks of age, as well as of symptomatic Mecp2^loxB/y/CreER (3) mice injected with Tam at 5 weeks of age (D). Tam-injected Mecp2^loxJ/y or Mecp2^loxB/y (1) and Veh-injected Mecp2^loxJ/y/CreER or Mecp2^loxB/y/CreER (2) are control littermates. Actin served as loading control. One-way ANOVA followed by appropriate post hoc for multiple-comparison tests were used to determine differences between groups. E, Quantitative RT-PCR analysis showing significant reduction in the level of Mecp2 mRNA in whole brain extracts of symptomatic 5W-Tam-injected Mecp2^loxJ/y/CreER mice but no significant differences in the levels of the indicated mRNAs encoding the specific synaptic proteins when compared with the mRNA levels in brains of Veh-injected Mecp2^loxJ/y/CreER control littermates. t test was used to compare within Veh and Tam groups. Error bars are mean ± SEM. *p < 0.05. n = 3 mice per genotype.