Project Summary The goal of this R21 project is to test whether enhancement of endogenous CREB (cAMP response element binding protein) signaling ameliorates disease phenotypes in a mouse model of the autism spectrum disorder, Rett syndrome (RTT). CREB is an evolutionarily conserved transcription factor that executes critical roles in metabolism, neuronal synaptic transmission, and cell growth regulation. Upregulation of CREB signaling has been linked to cancer and metabolic disease whereas reductions in CREB signaling are associated with age- dependent cognitive decline and a host of neurodegenerative disorders, including Alzheimer’s Disease, Huntington’s Disease and, of particular relevance to this proposal, RTT. CREB is activated by the second messenger cAMP through a two-hit mechanism involving its phosphorylation on S133 by protein kinase A (PKA), which recruits the transcriptional coactivator CREB-binding protein (CBP), and PKA-dependent nuclear import of CRTC proteins (cAMP/Ca2+-regulated transcriptional coactivators), which stabilize CREB-DNA interactions. We recently discovered that the critical S133 residue is dephosphorylated by protein phosphatase 2A (PP2A), which is recruited to CREB through short linear motifs (SLiMs) that are recognized by B56-type PP2A targeting subunits. Mutation of B56 binding sites in CREB strongly potentiated basal and stimulus dependent S133 phosphorylation and CREB transcriptional potential, informing a strategy for the genetic enhancement of CREB signaling in vivo. To this end, we used CRISPR/CAS9 to introduce a conservative E153D mutation that abolished B56-PP2A binding into the mouse Creb gene. Cells from homozygous CrebE153D mice exhibited increased S133 phosphorylation and upregulation of CREB-dependent gene expression, supporting further study of CrebE153D mice as a model for hypermorphic CREB signaling. In this study we will test whether CREB hyperactivation can reverse behavioral defects in a mouse model of RTT, a devastating neurodevelopmental disordered caused by X-linked mutations in the transcriptional repressor methyl-CpG binding protein (MeCP2). Previous work from the Chang laboratory revealed that CREB expression and S133 phosphorylation were downregulated in Mecp2- mutant neurons and that pharmacologic activators of CREB signaling partially reversed behavioral defects in Mecp2+/- mice. These findings set the stage for this proposal where we will use CrebE153D mice to test whether enhancement of endogenous CREB activity is sufficient for behavioral rescue in the RTT mouse model. The objectives of the proposal are to: (i) test the effect of CREB hyperphosphorylation on disease progression in male and female Mecp2 knockout (KO) mice; and (ii) determine impacts of B56-PP2A-CREB signaling on neuronal gene expression. In addition to testing genetic interaction between Creb and Mecp2, these studies, will define physiologic implications of the PP2A-B56-CREB signaling axis and develop the CrebE153D mod...