Understanding the Pathogenic Mechanisms of Rett Syndrome

Title Understanding the pathogenic mechanisms of Rett syndrome Abstract Rett Syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in methyl-CpG binding protein 2 (MECP2) gene. It occurs primarily in heterozygous females and represents one of the leading causes of intellectual disability in women. Despite its seemingly simple genetic root and the significant research progress thus far, the molecular mechanisms underlying RTT pathogenesis, such as the delayed onset and progressive clinical course, remain poorly understood. This is partially because mechanistic studies in heterozygous females are confounded by random X-chromosome inactivation (XCI) and the resulting mosaic expression of X-linked genes at the cellular level. We addressed this challenge in the last funding cycle by developing an allelic series of knock-in mice where endogenous wild-type (WT) or mutant MeCP2 was tagged by Cre-dependent biotinylation, thus allowing the isolation of WT-expressing and mutant-expression nuclei from the same heterozygous female brain. This strategy effectively circumvents the X-linked cellular mosaicism in heterozygous females but also overcomes cell type heterogeneity in the brain. Using this strategy, we completed a series of transcriptome profiling longitudinally across different stages of disease progression in a RTT relevant heterozygous female mouse model. We found that beginning soon after the developmental increase in non-CG DNA methylation (mCA) in early life, MeCP2-mutant neurons gradually accumulate small- magnitude changes in gene expression over time. In contrast, neighboring wild-type neurons are not affected. These findings raise new outstanding questions about 1) the molecular trigger that elicit more changes in gene expression over time; 2) the changes to the abundance of mRNAs that localize to specific cellular compartments; and 3) the origin of synaptic deficits at the level of individual synapses. We propose to address these questions with new and innovative genetic and genomic tools, including spatial transcriptomics, in this application. Our goal is to uncover pathogenic mechanisms of RTT and gain insights into the molecular and cellular control of neuronal gene expression and neural development in general.