Elsevier

Neuroscience

Volume 139, Issue 4, 2006, Pages 1449-1460
Neuroscience

Neuroanatomy
Temporal shift in methyl-CpG binding protein 2 expression in a mouse model of Rett syndrome

https://doi.org/10.1016/j.neuroscience.2006.01.060Get rights and content

Abstract

Rett syndrome is an X-linked neurodevelopmental disorder caused by mutations in methyl-CpG binding protein 2. Females with identical mutations in the methyl-CpG binding protein 2 gene can display varying severity of symptoms, suggesting that other factors such as X-chromosome inactivation affect phenotypic expression in Rett syndrome. Although X-chromosome inactivation is random and balanced in the blood and brain of the majority of girls with classic Rett syndrome, skewing in the ratio of expression of the mutant methyl-CpG binding protein 2-X to the wildtype-X affects the severity of symptoms. In this study, the pattern of immunostaining for methyl-CpG binding protein 2 was compared with that of neuronal nuclei specific protein, a pan-neuronal marker, to assess X-chromosome inactivation in a Rett syndrome mouse model. The number of cortical neurons and cortical volume were assessed by unbiased stereological measurements in younger adult (7–9 week old) wildtype (wildtype/methyl-CpG binding protein 2+/+), female heterozygous (heterozygous/methyl-CpG binding protein 2+/−), and null (methyl-CpG binding protein 2−/y) male mice and in older adult (24–95 week old) wildtype and heterozygous mice. The results showed that the number of neuronal nuclei specific protein–positive cells and cortical volume did not differ by genotype or age. However, younger adult heterozygous mice had significantly fewer methyl-CpG binding protein 2 cells and the pattern of methyl-CpG binding protein 2 staining was less distinct than in younger adult wildtype mice. However, in older adult heterozygous mice, the number and pattern of methyl-CpG binding protein 2-expressing neurons were similar to the wildtype. The ratio of methyl-CpG binding protein 2 to neuronal nuclei specific protein–stained neurons, a potential measure of X-chromosome inactivation, was close to 50% in the younger adult heterozygous mice, but nearly 70% in the older adult heterozygous mice. These results suggest that X-chromosome inactivation status changes with age. Such a change may underlie the more stable neurological function in older Rett syndrome patients.

Section snippets

Experimental procedures

Mecp2tm1.1Bird mice (Jackson Laboratory, Bar Harbor, ME, USA) on a C57BL/6 background (heterozygote backcrossed with C57BL/6 males for at least nine generations) were used for all experimental procedures. The Johns Hopkins University Institutional Animal Care and Use Committee approved all animal protocols, and guidelines from the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals were followed. All efforts were made to minimize the number of animals used and

MeCP2 immunostaining in the cortex of younger adult WT and HET mice differs in pattern and in neuronal number

MeCP2 immunostaining in younger adult (7–9 week old) WT controls exhibited distinct laminar boundaries (Figs. 2a and 3a–b). Adjacent sections labeled with the neuronal marker, NeuN had a very similar staining pattern (Fig. 2b). This similar pattern indicated that the anti-MeCP2 and anti-NeuN antibodies labeled the same neuronal populations in WT mice, as has previously been shown (Akbarian et al 2001, Shahbazian et al 2002b, Jung et al 2003, Mullaney et al 2004). Interestingly, the NeuN

Discussion

In this study we investigated the differences between younger (7–9 week old) and older (24–95 week old) adult WT, HET, and MECP2-null mice. We found morphological and corresponding numerical differences in the percentage of cells expressing MeCP2. Furthermore, in analyzing the two developmental stages, we found that the percentage of cortical neurons expressing MeCP2 increased from younger to older adult ages in HET animals.

The use of unbiased stereologic procedures allowed us to investigate

Acknowledgments

This work was supported by NICHD grants HD24448 and HD24061. We thank Ms. Pingping Zhang and Ms. Brandy McKinney for technical assistance and Dr. SakkuBai Naidu and Dr. Walter Kaufmann for their helpful suggestions on the manuscript.

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