Generation of Genetic Protein Synthesis Knockdown Mice

众所周知，当动物服用蛋白质合成抑制剂（如大霉素）时，这些动物就会失去长期记忆。大霉素会阻止动物大脑中蛋白质的合成。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，之后它们不再受海马体操纵的影响。这个过程被称为“系统巩固”，通过这个过程，记忆可能会从海马体转移到大脑皮层。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，海马体再次参与。抑制蛋白质合成也被证明破坏突触可塑性，甚至在几个不同的系统的感觉表征。越来越多的证据表明，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物质，如大霉素、艾美汀和环己亚胺来抑制蛋白质合成。最近的研究表明，一种蛋白质合成化学抑制剂诱导mRNA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，在哺乳动物细胞中，已发现大霉素可激活MAP激酶通路。为了克服这些缺点，在活体动物中诱导蛋白质合成敲低的基因操作已经被期望用于细胞和系统水平的记忆巩固研究。

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.