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.