Generation of Genetic Protein Synthesis Knockdown Mice

众所周知，当动物被蛋白质合成抑制剂治疗时，这些动物会失去长期记忆，这些蛋白质合成抑制剂会阻止动物大脑中蛋白质的产生。这一观察结果使我们预测，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，之后它们不再容易受到海马体操作的影响。这一过程被称为“系统巩固”，即记忆大概是从海马体转移到大脑皮层的过程。然而，最近的研究表明，在完成最初的细胞巩固过程后，记忆在回忆时再次与海马体接触。蛋白质合成的抑制也被证明扰乱了突触的可塑性，甚至在几个不同的系统中的感觉表达。这些不断积累的证据表明，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，大多数研究都使用了像茴香素、埃米汀和放线菌亚胺这样的化学物质来抑制蛋白质合成。最近的研究表明，一种蛋白质合成的化学抑制剂可以诱导mRNA的表达，这一过程被称为超诱导。它可以通过三种方式之一发生，包括(I)mRNA稳定，(Ii)激活细胞内信号通路，或(Iii)干扰转录下调。此外，茴香素已被证明可以激活哺乳动物细胞中的MAPK通路。为了克服这些缺陷，在活体动物中对蛋白质合成敲除进行可诱导的遗传操作，已被期望在细胞和系统水平上研究记忆巩固。 自2003年10月以来，我们开始了一个项目，开发可诱导的基因抑制小鼠大脑中的蛋白质合成。众所周知，依赖于RNA的双链蛋白激酶R(PKR)通过磷酸化eIF2α来抑制细胞中大多数蛋白质的合成，eIF2α是蛋白质翻译过程中启动多肽延伸的关键因素。PKR结构域的二聚化是激酶激活所必需的，并在给药后被诱导；为了利用这一过程，我们使用了一个化学诱导的二聚化系统，基于FKBP12的系统，来控制PKR的活性。姜志红在巨细胞病毒(CMV)启动子的控制下构建了HA-FKBP-PKR的cDNA载体，并将其导入SH-SY5Y细胞。24小时后，化学交联剂AP20187(ARIAD PharmPharmticals，Inc.)被加入以诱导PKR的二聚化以激活其。AP20187处理16h后，观察AP20187对细胞从头合成的抑制作用，并用几种抗体检测HA-Tag、FKBP、eIF2a及其在蛋白凝胶电泳图上的磷酸化形式。接下来，她与Jim Pickel博士(转基因核心设备)合作，将α-CaM Kinase II启动子控制下的loxP-LacZ-loxP-FKBP-PKR构建载体注射到小鼠卵子中，创建了转基因小鼠；其中几个品系显示LacZ在小鼠前脑中高表达。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注射AP20187后，小鼠大脑中是否有效地发生了从头蛋白合成的抑制。此外，评估大脑蛋白质的从头合成受到多大程度的抑制也将是至关重要的。差异凝胶电泳(DGE)是一种蛋白质组学工具，可以对数千种蛋白质进行分离和定量。我们计划使用DGE技术来评估AP20187治疗后转基因小鼠大脑中蛋白质合成受到多大程度的抑制。 在另一个单独的项目(MH002824-03)中，我们正在使用BAC转基因技术创建海马区CA1限制性Cre转基因系。一旦CA1-Cre系建立，FKBP-PKR与CA1-Cre系互交的双条件转基因系将是研究海马区依赖记忆系统巩固的一个很好的遗传工具。

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.