A Drosophila Model of Neuronal MECP2 Function

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Methyl-CpG binding protein 2 (MECP2) is a widely abundant, multifunctional regulator of gene expression in the CNS. In humans, both loss- and gain-of-function mutations of MECP2 cause mental retardation and motor dysfunction classified as either Rett Syndrome (RTT, loss-of-function) or MECP2 Duplication Syndrome (MDS, gain-of-function). There are currently no effective treatments for these conditions. While Mecp2 is classically known to repress transcription by binding to methylated DNA, many additional gene regulatory functions have been identified. At the cellular level, mis-regulation of Mecp2 leads to synaptic and dendritic defects. Research on Mecp2 function in neurons has relied primarily on mouse models of Mecp2 deletion, mutation, and over-expression, but large-scale screening and in vivo validation in the mouse model is time consuming.The objective of this proposal is to use Drosophila melanogaster as a model system to study specific cellular and molecular functions of MECP2 gain-of-function in neurons. The Drosophila genome is sparsely methylated and therefore provides a unique opportunity to specifically examine non-methyl DNA binding functions of hMECP2. We have shown that expression of human MECP2 (hMECP2) in Drosophila motoneurons leads to distinct defects in dendritic structure and motor behavior, as reported with Mecp2 gain-of-function in humans and mice. These effects are dependent on specific MECP2 protein domains and can be rescued by genetic interaction experiments; hence, they are caused by gain of hMECP2 function and not general toxicity. We used RNA-sequencing technology to identify 45 genes and 120 transcript isoforms differentially regulated with hMECP2 expression in Drosophila. Kibra, a gene associated with learning and memory in humans, was identified as a top target from this screen. Kibra also activates the Hippo kinase cascade (HKC), a pathway involved in the regulation of dendritic growth. Pilot studies indicate that knockdown of kibra together with co-expression of hMECP2 rescue the dendritic defects. The first aim will test the hypothesis that MECP2-induced dendritic and behavioral defects depend on up-regulation of kibra. We will confirm quantitatively if knockdown of kibra can rescue the dendritic and behavioral effects of hMECP2 expression in Drosophila. We will additionally conduct experiments in mouse primary neuron cultures to determine whether these mechanisms are conserved in mammalian neurons. The second aim will test the hypothesis that hMECP2 increases activation of the HKC, and that this activation mediates the dendritic and behavioral defects observed with hMECP2 expression. Investigating these novel MECP2 targets identified in Drosophila can help elucidate how disruption of MeCP2 leads to nervous system and behavioral defects and can ultimately lead to future therapeutic strategies for RTT and MDS.