Expression of the methyl-CpG-binding protein MeCP2 in rat brain. An ontogenetic study

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Abstract

Rett syndrome (RS) is caused by mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2) and is characterized by arrested postnatal neurodevelopment. We followed the expression of MeCP2 protein in various brain structures of normal rat from birth to 2 years of age. By measuring the amount of protein using the Western blot technique, or by determining the percentage of immunoreactive cells, significant heterogeneity in MeCP2 distribution among various brain areas was observed. Highest expression was found in olfactory bulb and in frontal cortex. In contrast, little expression was detected in caudate-putamen, septum and hippocampus. Except in the olfactive nuclei, very few cells showed detectable MeCP2 protein at birth. The number increased during the first week of age, especially in cortex and nucleus accumbens. Rather than playing a global role in gene transcription, the heterogeneous distribution of MeCP2 transcription factor favors the idea that it has a specialized function in neurons.

Introduction

The classical form of Rett syndrome (RS), found almost exclusively in females, is a disorder of arrested neuronal development Armstrong, 2001, Kerr, 2002, Percy, 2002. Affected girls are born healthy, appear to develop normally until age 6–18 months, achieving expected motor and language skills (Hagberg et al., 1983). Their neurological development is then arrested and begins regressing in a predictable pattern (Hagberg and Witt-Engerstrom, 1987). The disorder is characterized essentially by severe cognitive impairment, autistic behavior, stereotypic movements and seizures (Naidu, 1997). Functional deficits are associated with abnormal levels of neurotransmitters and peptides (Wenk, 1997). In most cases, the syndrome was found associated with mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2) Amir et al., 1999, Shahbazian and Zoghbi, 2002. Once bound to methylated DNA, MeCP2 is thought to silence transcription of downstream genes by virtue of its interaction with a histone deacetylase/Sin3 complex Jones et al., 1998, Nan et al., 1997. To understand the molecular basis of RS, it is essential to identify the target genes of MeCP2 repression. Unfortunately, a recent report shows that brains of MeCP2 mutant mice have few, if any, genes that are significantly changed in their level of expression (Tudor et al., 2002).

Three MeCP2 transcripts are found at varying levels in most tissues examined, with no obvious preference for nervous tissue, except that the brain displays preferentially a 10-kb transcript compared with other transcripts (Reichwald et al., 2000). Few data are available on the expression of MeCP2 protein in the central nervous system. Unlike the mRNA, the protein is expressed at higher levels in the brain than in many other tissues LaSalle et al., 2001, Shahbazian et al., 2002b. Within the brain, MeCP2 is found in neurons but is not expressed to detectable levels in differentiated astrocytes Akbarian et al., 2001, Jung et al., 2003. Here, we studied the expression of MeCP2 protein in various brain structures from normal rat. Several cortical areas with major functional implication were selected, in addition to the septal nuclei and the hippocampus that may be involved in the cognitive deficits observed in RS, and the striatum that was also suggested to be implicated in the disease Armstrong, 2001, Kerr, 2002. Since RS is characterized as a disease of arrested development after birth, we followed the protein expression throughout rat life, from birth to old age corresponding to 112 weeks.

Section snippets

Animals and tissue preparation

Female Long–Evans rats (Janvier, France) were housed with a fixed 12-h light–dark cycle and free access to food and water. All procedures involving animal care were conducted in conformity with institutional guidelines, in compliance with current laws and policies (council directive 87848, Service Vétérinaire de la Santé et de la Protection animales). Animals were sacrificed by an overdose of pentobarbital (100 mg/kg, ip) and perfused transcardially with saline followed by 4% formaldehyde in

Results

We studied MeCP2 protein expression in several brain structures of normal Long–Evans rat from birth to 2 years of age. Fig. 2 illustrates MeCP2 immunohistochemistry of cells from selected areas: the olfactory nuclei, frontal cortex, dorsal caudate and CA2/CA3 region of dorsal hippocampus of newborn rats (taken during the 5-h postnatal period) and rats sacrificed at various ages. A very large majority of cells displayed MeCP2 immunoreactivity exclusively in the nucleus, in agreement with the

Discussion

The present ontogenetic study on MeCP2 distribution demonstrates a very heterogeneous distribution of MeCP2 protein in normal rat brain, as assessed by measuring the amount of protein as well as percentages of immunoreactive cells. It has to be kept in mind, however, when interpreting the latter data, that MeCP2 is found primarily in neurons and not in glia (Akbarian et al., 2001). In effect, it was not detected in rat astrocytes (Jung et al., 2003), whereas another study reported some low

Acknowledgements

We thank Dr. P. Jouvert and A. Lazaris for help with immunohistological techniques, and Dr. K. Langley for critical reading of the manuscript.

References (24)

  • D. Balmer et al.

    Elevated methyl-CpG-binding protein 2 expression is acquired during postnatal human brain development and is correlated with alternative polyadenylation

    J. Mol. Med.

    (2003)
  • R.Z. Chen et al.

    Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice

    Nat. Genet.

    (2001)
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