Generation of Genetic Protein Synthesis Knockdown Mice

遗传蛋白质合成敲低小鼠的产生

• 批准号: 7137895
• 负责人: Kazutoshi Nakazawa
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7137895
• 关键词: behavior test; behavioral /social science research tag; behavioral genetics; biotechnology; brain metabolism; calmodulin dependent protein kinase; crosslink; cytomegalovirus; dimer; enzyme induction /repression; gel electrophoresis; genetic promoter element; genetically modified animals; hippocampus; laboratory mouse; long term memory; memory disorders; neuropsychology; protein biosynthesis; protein kinase; transfection /expression vector

It has been well known that when animals are treated with protein synthesis inhibitors, such as anisomycin, which stop the production of proteins in the animals' brains, these animals lose their long-term memory. This observation has led us to predict that the formation of long-term memory requires new protein synthesis. Furthermore, certain types of memories are dependent on the hippocampus for a short period of time following training, after which they are no longer susceptible to hippocampal manipulations. This process has been called "systems consolidation", a process by which memory is presumably transferred from hippocampus to cortex. However, recent studies have suggested that, after having completed the initial cellular consolidation process, a memory once again engages the hippocampus when recalled. Inhibition of protein synthesis has also been shown to disrupt synaptic plasticity and even sensory representation in several different systems. These accumulating evidence has suggested that continued protein synthesis is essential for the normal function of the brain. The caveat of this research is that most of the studies use chemicals such as anisomycin, emetine, and cycloheximide to inhibit protein synthesis. Recent studies have demonstrated that a protein synthesis chemical inhibitor induces mRNA expression, a process called super-induction. It can occur in one of the three ways, including (i) mRNA stabilization, (ii) activation of intracellular signaling pathways, or (iii) interference with transcriptional down-regulation. In addition anisomycin has been shown to activate MAP kinase pathway in mammalian cells. In order to overcome these drawbacks, inducible genetic manipulation of protein synthesis knockdown in the live animals has been desired for the study of memory consolidation at both a cellular and systems level. Since October 2003, we have started a project to develop inducible genetic suppression of protein synthesis in the mouse brain. It is known that double-strand RNA-dependent protein kinase R (PKR) inhibits synthesis of most proteins in a cell by phosphorylating eIF2 alpha, a key factor to initiate peptide elongation during protein translation process. Dimerization of the PKR domain is required for kinase activation and induced upon a drug administration; to take advantage of this process, we used a chemical induced dimerization system, FKBP12-based system, to control the activity of PKR. Zhihong Jiang created a cDNA construct of HA-FKBP-PKR under the control of cytomegalovirus (CMV) promoter was prepared and transfected into SH-SY5Y cells. Twenty-four hours later a chemical cross-linking inducer, AP20187 (ARIAD Pharmaceuticals, Inc.) was added to induce the dimerization of PKR for its activation. De novo protein synthesis inhibition was observed 16 hours after AP20187 treatment, which was evaluated by using several antibodies to detect HA-tag, FKBP, eIF2a and its phosphorylation form on the protein gel electrophoresis. Next, in collaboration with Dr. Jim Pickel (Transgenic Core Facility), she injected the construct of loxP-LacZ-loxP-FKBP-PKR under the control of alpha CaM Kinase II promoter, into mouse eggs to create transgenic mice; several of these lines show high expression of LacZ in the mouse forebrain. The key issue to conduct mouse behavioral testing using this inducible system is to ask whether de novo protein synthesis inhibition efficiently occurs in the mouse brain following intra-peritoneal administration of AP20187. Also, it will be critical to evaluate to what extent de novo protein synthesis is inhibited among brain proteins. Difference gel electrophoresis (DiGE) is a proteomics tool that permits the separation and quantification of thousands of proteins. We plan to use a DiGE technology to evaluate to what extent the protein synthesis is overall inhibited in the transgenic mouse brain following AP20187 treatment. In a separate project (MH002824-03), we are working to create hippocampal CA1-restricted Cre transgenic lines using BAC transgenic technology. Once the CA1-Cre line is established, the double conditional transgenic line inter-crossed between FKBP-PKR and CA1-Cre line, would be a great genetic tool for the study of systems consolidation of hippocampus-dependent memory.

众所周知，当动物用蛋白质合成抑制剂治疗时，如茴香霉素，它会停止动物大脑中蛋白质的产生，这些动物会失去长期记忆。这一观察使我们预测，长期记忆的形成需要新的蛋白质合成。此外，某些类型的记忆在训练后的短时间内依赖于海马体，之后它们不再受海马体操纵的影响。这个过程被称为“系统整合”，这是一个记忆从海马体转移到皮层的过程。然而，最近的研究表明，在完成了最初的细胞巩固过程后，当回忆起记忆时，海马体会再次参与其中。蛋白质合成的抑制也被证明会破坏突触可塑性，甚至在几个不同的系统中的感觉表征。这些累积的证据表明，持续的蛋白质合成对大脑的正常功能至关重要。这项研究的警告是，大多数研究使用化学品，如茴香霉素，emphetamine和放线菌酮来抑制蛋白质合成。最近的研究表明，蛋白质合成化学抑制剂诱导mRNA表达，这一过程称为超诱导。它可以以三种方式之一发生，包括（i）mRNA稳定，（ii）细胞内信号传导途径的激活，或（iii）干扰转录下调。此外，茴香霉素已显示激活哺乳动物细胞中的MAP激酶途径。为了克服这些缺点，在活体动物中蛋白质合成敲低的诱导型遗传操作已被期望用于在细胞和系统水平上研究记忆巩固。 自2003年10月以来，我们已经开始了一个项目，以开发诱导基因抑制蛋白质合成的小鼠大脑。已知双链RNA依赖性蛋白激酶R（PKR）通过磷酸化eIF 2 α来抑制细胞中大多数蛋白质的合成，eIF 2 α是蛋白质翻译过程中启动肽延伸的关键因子。PKR结构域的二聚化是激酶激活所需的，并在给药后诱导;为了利用这一过程，我们使用了化学诱导的二聚化系统，基于FKBP 12的系统，以控制PKR的活性。构建了巨细胞病毒（CMV）启动子调控下的HA-FKBP-PKR基因的cDNA构建体，并转染SH-SY 5 Y细胞。24小时后，将化学交联诱导剂AP 20187（ARIAD Pharmaceuticals，Inc.）以诱导PKR的二聚化以使其活化。在AP 20187处理后16小时观察到从头蛋白合成抑制，通过使用几种抗体在蛋白凝胶电泳上检测HA标签、FKBP、eIF 2a及其磷酸化形式进行评价。接下来，她与Jim Pickel博士（转基因核心设施）合作，将α CaM激酶II启动子控制下的loxP-LacZ-loxP-FKBP-PKR构建体注射到小鼠卵中以创建转基因小鼠;其中几个品系显示LacZ在小鼠前脑中的高表达。使用这种诱导系统进行小鼠行为测试的关键问题是询问在腹膜内给予AP 20187后小鼠大脑中是否有效地发生从头蛋白质合成抑制。此外，这将是至关重要的，以评估在何种程度上从头蛋白质合成抑制脑蛋白质。差异凝胶电泳（DiGE）是一种蛋白质组学工具，可以分离和定量数千种蛋白质。我们计划使用DiGE技术来评估在AP 20187处理后转基因小鼠脑中蛋白质合成被整体抑制的程度。 在一个单独的项目（MH 002824 -03）中，我们正在使用BAC转基因技术创建海马CA 1限制性Cre转基因系。一旦获得CA 1-Cre系，FKBP-PKR与CA 1-Cre系互交获得的双条件转基因系，将为研究海马依赖性记忆的系统巩固提供重要的遗传工具。