Genetic enhancement of CREB signaling in Rett Syndrome

Rett 综合征中 CREB 信号传导的遗传增强

基本信息

批准号：
10227232
负责人：
Randal Scot Tibbetts
金额：
$ 19.44万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10227232
关键词：
Aging Alleles Alzheimer&apos s Disease Behavior Behavioral Binding Binding Proteins Binding Sites CREBBP gene CRISPR/Cas technology Cells ChIP-seq Complex Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cyclic AMP-Responsive DNA-Binding Protein DNA Binding DNA-Binding Proteins Defect Disease Disease Progression Exhibits Female Gene Expression Gene Expression Profiling Gene Mutation Gene Proteins Genes Genetic Enhancement Genetic Transcription Goals Huntington Disease Hyperactivity Impaired cognition Induced pluripotent stem cell derived neurons Knock-in Mouse Knockout Mice Laboratories Link Malignant Neoplasms Mediating Memory Metabolic Diseases Metabolism Methyl-CpG-Binding Protein 2 Modeling Molecular Mouse Strains Mus Mutant Strains Mice Mutation Neurodegenerative Disorders Neurodevelopmental Disorder Neurons Nuclear Import Pharmacology Phosphodiesterase Inhibitors Phosphorylation Physiological Play Pluripotent Stem Cells Protein Kinase Protein Subunits Protein phosphatase Proteins Regulation Reporting Research Rett Syndrome Role Second Messenger Systems Signal Transduction Signaling Protein Stimulus Synaptic Transmission Testing Therapeutic Transcription Coactivator Transcription Repressor Up-Regulation Work age related attenuation autism spectrum disorder behavioral study causal variant cell growth cell growth regulation disease phenotype experimental study gene function genetic approach genetic testing genome-wide in vivo insight loss of function male mouse model mutant nervous system disorder neurophysiology novel protein expression protein function recruit therapeutic target tool transcription factor tumorigenesis

项目摘要

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.

项目摘要这个R21项目的目的是测试内源性CREB（营地响应元件）是否增强结合蛋白）信号传导在自闭症谱系障碍的小鼠模型中改善疾病表型， RETT综合征（RTT）。 CREB是一种进化配置的转录因子，在代谢，神经元突触传递和细胞生长调节。 CREB信号的上调具有我们与癌症和代谢疾病有关，而CREB信号的降低与年龄有关依赖的认知能力下降和许多神经退行性疾病，包括阿尔茨海默氏病，亨廷顿氏病，与该提案特别相关，RTT。 Creb被第二次激活通过蛋白激酶A（PKA）在S133上的磷酸化的两次打击机制，通过两次打击的机制，Messenger Camp，收益转录共激活因子CREB结合蛋白（CBP）和PKA依赖性核进口 CRTC蛋白（CAMP/CA2+调节的转录共激活剂）的构成，可稳定CREB-DNA相互作用。我们最近发现，临界S133居住地被蛋白质磷酸酶2a（PP2A），磷酸化。通过B56型PP2A靶向识别的短线性图案（Slim）招募到CREB 亚基。 CREB中B56结合位点的突变，依赖于S133的基本和刺激的突变磷酸化和CREB转录潜力，为CREB遗传增强的策略提供了信息体内信号传导。为此，我们使用CRISPR/CAS9引入了废除的保守E153D突变 B56-PP2A结合到小鼠Creb基因中。来自纯合Crebe153d小鼠暴露的细胞增加了S133 CREB依赖性基因表达的磷酸化和上调，支持CREBE153D的进一步研究小鼠作为甲状腺高晶状体信号传导的模型。在这项研究中，我们将测试CREB过度激活是否可以逆转小鼠模型中的行为缺陷 RTT，一种由转录代表中X连锁突变引起的毁灭性神经发育障碍甲基-CPG结合蛋白（MECP2）。 Chang Laboratory的先前工作表明CREB表达在MECP2突变神经元中下调了S133磷酸化，并将其药物激活剂的药物激活剂 CREB信号传导部分逆转了MECP2 +/-小鼠的行为缺陷。这些发现为此奠定了基础提案我们将在其中使用Crebe153d小鼠测试内源性CREB活性的增强是否为足以在RTT小鼠模型中进行行为救援。该提案的目标是：（i）测试效果男性和雌性MECP2敲除（KO）小鼠疾病进展的CREB高磷酸化；（ii）确定B56-PP2A-CREB信号传导对神经元基因表达的影响。除了测试通用 CREB和MECP2之间的相互作用，这些研究将定义PP2A-B56-CREB的生理意义信号轴并开发CREBE153D模型作为操纵其他内源性CREB信号的工具生理范例。