Quantitative localization of heterogeneous methyl-CpG-binding protein 2 (MeCP2) expression phenotypes in normal and Rett syndrome brain by laser scanning cytometry

Abstract

Rett syndrome (RTT) is an X-linked, dominant neurodevelopmental disorder caused by mutations in MECP2, encoding the methyl-CpG-binding protein 2 (MeCP2). A major paradox in the pathogenesis of RTT is how mutations in ubiquitously transcribed MECP2 result in a phenotype specific to the central nervous system (CNS) during postnatal development. To address this question, we have used a novel approach for quantitating the level and distribution of wild-type and mutant MeCP2 in situ by immunofluorescence and laser scanning cytometry. Surprisingly, cellular heterogeneity in MeCP2 expression level was observed in normal brain with a subpopulation of cells exhibiting high expression (MeCP2(hi)) and the remainder exhibiting low expression (MeCP2(lo)). MeCP2 expression was significantly higher in CNS compared with non-CNS tissues of human and mouse by automated quantitation of MeCP2 on multiple tissue arrays. Quantitative localization of MeCP2 expression phenotypes in normal human brain showed a mosaic, but distinct, distribution pattern, with MeCP2(hi) neurons highest in layer IV of the cerebrum and MeCP2(lo )neurons highest in the granular layer of the cerebellum. In female RTT brains, MECP2 mutant-expressing cells were identified as cells negative for the MeCP2 C-terminal epitope. MECP2 mutant-expressing cells were randomly localized in Rett cerebrum and cerebellum and showed normal MeCP2 expression with N-terminal-specific anti-MeCP2. These results demonstrate a CNS-specific cellular phenotype of MeCP2 high expression and suggest that MECP2 mutations in RTT are only manifested in MeCP2(hi) cells. In addition, our results demonstrate the power of laser scanning cytometry in examining complex cellular phenotypes in disease pathogenesis.

Separate Respiratory Phenotypes in Methyl-CpG-Binding Protein 2 (Mecp2) Deficient Mice

“…Measurements of carbon dioxide levels in RTT patients during episodes of hyperventilation show significant hypocapnia before apnea (6,8). Mecp2 is heavily expressed in human (17) and mouse lung (18). We hypothesized that differences in lung volume and respiratory pattern contribute to respiratory depression and apnea in RTT.…”

Rett Syndrome: Reaching for Clinical Trials

“…One factor that has not received major attention is the clonal distribution of normal and mutant X chromosomes in brain as differences in expression patterns may lead to markedly different clinical phenotypes. LaSalle et al noted this in 2001 [45], but scarce mention has been made in recent years. Attempts at identifying variations in XCI in blood have indicated skewing in > 25% of RTT individuals, which is approximately double the occurrence in the general population.…”

Histone modifications in Rett syndrome lymphocytes: a preliminary evaluation

“…For detection and quantification of MeCP2 immunoreactivity, we used polyclonal antibodies (Abs) targeting either the N-or C-terminus of the protein [36]: an Ab directed to residues 9-27 of human MeCP2, kindly provided by Dr John Christodoulou (Children's Hospital at Westmead, Sydney, Australia), and an Ab that targets a highly conserved Cterminal epitope (i.e. residues 465-478) of mouse MeCP2 (Upstate Biotechnology, Waltham, MA, USA) [36][37][38]. Non-acetylated (also termed total) [17] and acetylated (Ac) H3 and H4, and di-methylated (Me) H3 were also detected by Abs supplied by Upstate.…”