Genetic enhancement of CREB signaling in Rett Syndrome

项目摘要 该R21项目的目标是测试内源性CREB(cAMP反应元件)的增强 结合蛋白)信号可改善自闭症谱系障碍小鼠模型的疾病表型， Rett综合征(RTT)。CREB是一种进化上保守的转录因子，在 代谢、神经元突触传递和细胞生长调节。CREB信号的上调有 与癌症和代谢性疾病有关，而CREB信号的减少与年龄有关- 依赖性认知能力下降和一系列神经退行性疾病，包括阿尔茨海默病， 亨廷顿氏病，以及与这一提议特别相关的RTT。CREB由第二个激活 信使cAMP通过蛋白激酶A(PKA)在S133上磷酸化的两次作用机制， 它招募了转录共激活物CREB结合蛋白(CBP)，以及依赖PKA的核进口 CRTC蛋白(cAMP/Ca~(2+)调节的转录共激活因子)，稳定CREB-DNA相互作用。 我们最近发现，关键的S133残基被蛋白磷酸酶2A(PP2A)去磷酸化， 它通过B56型PP2A靶向识别的短线性基序(SLIMs)招募到CREB 亚单位。CREB中B56结合位点突变强烈增强基础和刺激依赖性S133 磷酸化和CREB转录潜能，为CREB的遗传增强策略提供信息 体内的信号。为此，我们使用CRISPR/Cas9引入了一个保守的E153D突变，该突变取消了 B56-PP2A与小鼠Creb基因的结合。来自纯合子CrebE153D小鼠的细胞显示S133增加 CREB依赖的基因表达的磷酸化和上调，支持对CrebE153D的进一步研究 小鼠作为高形态CREB信号的模型。 在这项研究中，我们将测试CREB过度激活是否可以逆转小鼠的行为缺陷 RTT，一种由转录抑制因子X连锁突变引起的破坏性神经发育障碍 甲基-CpG结合蛋白(MeCP2)。张实验室之前的工作表明，CREB表达 和S133的磷酸化水平在MeCP2突变的神经元中下调，而药物激活剂 CREB信号部分逆转了MeCP2+/-小鼠的行为缺陷。这些发现为这一点奠定了基础 我们将使用CrebE153D小鼠来测试内源性CREB活性的增强是否 足以在RTT小鼠模型中进行行为救援。这项建议的目的是：(一)测试效果 CREB过度磷酸化对雄性和雌性MeCP2基因敲除(KO)小鼠疾病进展的影响；以及(Ii) 确定B56-PP2A-CREB信号对神经元基因表达的影响。除了测试基因 这些研究将确定PP2A-B56-CREB在CREB和CREB之间的相互作用 并开发了CrebE153D模型，作为操纵其他植物内源CREB信号的工具 生理学范式。

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 model as a tool for manipulating endogenous CREB signaling in other physiologic paradigms.