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J Neurosci. 2012 Jul 18;32(29):10021-34. doi: 10.1523/JNEUROSCI.1316-12.2012.

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.

Nguyen MV, Du F, Felice CA, Shan X, Nigam A, Mandel G, Robinson JK, Ballas N.

Source

Department of Biochemistry, Stony Brook University, Stony Brook, New York 11794, USA.

Abstract

Mutations in the X-linked gene, methyl-CpG binding protein 2 (Mecp2), underlie a wide range of neuropsychiatric disorders, most commonly, Rett Syndrome (RTT), a severe autism spectrum disorder that affects approximately one in 10,000 female live births. Because mutations in the Mecp2 gene occur in the germ cells with onset of neurological symptoms occurring in early childhood, the role of MeCP2 has been ascribed to brain maturation at a specific developmental window. Here, we show similar kinetics of onset and progression of RTT-like symptoms in mice, including lethality, if MeCP2 is removed postnatally during the developmental stage that coincides with RTT onset, or adult stage. For the first time, we show that brains that lose MeCP2 at these two different stages are actively shrinking, resulting in higher than normal neuronal cell density. Furthermore, we show that mature dendritic arbors of pyramidal neurons are severely retracted and dendritic spine density is dramatically reduced. In addition, hippocampal astrocytes have significantly less complex ramified processes. These changes accompany a striking reduction in the levels of several synaptic proteins, including CaMKII α/β, AMPA, and NMDA receptors, and the synaptic vesicle proteins Vglut and Synapsin, which represent critical modifiers of synaptic function and dendritic arbor structure. Importantly, the mRNA levels of these synaptic proteins remains unchanged, suggesting that MeCP2 likely regulates these synaptic proteins post-transcriptionally, directly or indirectly. Our data suggest a crucial role for MeCP2 in post-transcriptional regulation of critical synaptic proteins involved in maintaining mature neuronal networks during late stages of postnatal brain development.

PMID:: 22815516; [PubMed - indexed for MEDLINE]
PMCID:: PMC3461266

Free PMC Article

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Figure 2

The loss of MeCP2 at late juvenile or adult stages induces RTT-like phenotypes in males and females

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). Red arrows indicate the time of Tam or Veh injection. 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 ± s.e.m. 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 ± s.e.m. t-test was used to compare within the 5W- and the 10W-Tam- injected groups. C, F, I, and L) Images illustrating the hind limb 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 over-grooming (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.

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

J Neurosci. 2012 July 18;32(29):10021-10034.

Figure 4

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 littermate, Mecp2^loxJ/y+T (loxJ/y+T), and Mecp2^loxJ/y/CreER+V (loxJ/y/Cre+V) mice. Brains are from16 weeks post 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. Red arrow represents the time of injection. Fragmented lines distinguish between the brains that are smaller (bellow) 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 post-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 post-injection) and smaller than the WT brains at 10 weeks of age and the 10W-Tam-injected Mecp2^loxJ/y/CreER 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 ± s.e.m. *P<0.05. n=4–6 mice per genotype. H–I) Time course analysis of 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.

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

J Neurosci. 2012 July 18;32(29):10021-10034.

Figure 6

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, 20 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 post 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 mice when compared to Veh-injected Mecp2^loxJ/y/CreER or Tam-injected wild-type (WT) mice, 18 weeks post 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 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 ± s.e.m. *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 post injection). n=3 mice per genotype and age. n=12–15 neurons per animal.

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

J Neurosci. 2012 July 18;32(29):10021-10034.

Figure 7

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 post 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 5W-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 post 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 mice when compared to Veh-injected Mecp2^loxJ/y/CreER or Tam-injected wild-type (WT) mice, 18 weeks post 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 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 ± s.e.m. *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 post injection). n=3 mice per genotype and age. n=12–15 neurons per animal.

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

J Neurosci. 2012 July 18;32(29):10021-10034.

Figure 10

Schematic model for the function of MeCP2 during critical stage of postnatal brain development and in the mature brain

A) During postnatal development, the size and weight of normal brain are increased while neurons mature as evident by the increase in dendritic complexity and synaptogenesis. The addition of synaptic proteins during late maturation increases synaptic strength, which in turn increases dendritic branching and stability of dendritic arbor structure. This process involves neuron:astrocyte interactions. B) In classic RTT caused by germline mutations in MeCP2, the brain is smaller than normal and neurons exhibit reduced dendritic arborization and synaptic protein expression. C-D) Inactivation of Mecp2 in otherwise normal mice during their late juvenile period of development (C) or at adult age (D) results in brain phenotypes typical for classic RTT. However, while the reduced brain size in late juvenile mice is caused partly by developmental stagnation and partly by shrinkage of the normally developed brain, in adult mice it is caused solely by shrinkage. In both cases, synaptic protein expression is reduced, and dendritic arbors of the already matured neurons are retracted. Similar to neurons, the morphology of astrocytes is also altered exhibiting fewer and shorter processes which together likely affect neuron:astrocyte interactions and therefore disruption of the mature neuronal networks. The loss of MeCP2, whether is due to germline mutations or induced at postnatal stages, during late developmental stage (juvenile stage) or adult stage, results in similar brain abnormalities, which lead to RTT phenotypes. E) In the reverse situation, global re-expression of MeCP2 in symptomatic RTT mice with germline mutation in Mecp2 results in reversal of RTT symptoms and neuronal function (Guy et al., 2007), and postnatal re-expression of MeCP2 in astrocytes inhibits the progression of RTT phenotypes and restores dendritic arbor structure and the level of at least the synaptic protein Vglut (Lioy et al., 2011).

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

J Neurosci. 2012 July 18;32(29):10021-10034.