Generation and Analysis of Genetic Protein Synthesis Kno

遗传蛋白质合成知识的产生与分析

• 批准号: 6982768
• 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/6982768
• 关键词: behavioral /social science research tag; brain cell; gene targeting; genetic crossing over; genetically modified animals; hippocampus; laboratory mouse; memory; phosphorylation; protein biosynthesis; protein kinase; transcription factor

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. This phenomenon is now called reconsolidation, which seems to be protein synthesis-dependent. Here a simple question arises as to whether memories reach a fixed stable state, or if they are subject to change every time they are activated. When the possibility is added that memories ?move? from one area of the brain to another after a certain period of time, we may call into question the conventional view of stable memory or its consolidation. The caveat of the research of this field is that most of the studies use chemicals like anisomycin to inhibit protein synthesis. Recent studies have demonstrated that a protein synthesis 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 last year, 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) phosphorylates eIF2a, a key enzyme to initiate peptide elongation during protein translation process, and inhibit synthesis of most proteins in a cell. Taking the advantage of inducible dimerization of PKR domain upon a drug administration, that is required for kinase activation, Dr. Zhihong Jiang prepared a cDNA construct of loxP-LacZ-loxP-FKBP-PKR under the control of Cam Kinase II promoter. The transgenic core facility (Dr. Jim Pickel) injected the construct into mouse eggs to create its transgenic mice, and she has obtained several lines which shows high expression of LacZ in the mouse forebrain. Once these lines are crossed with a particular Cre-recombinase transgenic line, such as CA1-Cre line, we expect to create CA1-restricted genetic protein synthesis knockdown mice in an inducible manner. In a separate project, she is also trying to create hippocampal CA1-restricted Cre transgenic lines using BAC transgenic technology. The double conditional transgenic line inter-crossed between FKBP-PKR and CA1-Cre would be quite beneficial for the study of system consolidation of hippocampus-dependent memory. The key point in this project is whether protein synthesis inhibition is sufficient to take place in a brain cell where Cre recombinase is highly expressed upon the drug administration. The experiment to test this issue is now underway.

众所周知，当动物接受蛋白质合成抑制剂（例如茴香霉素）治疗时，会阻止动物大脑中蛋白质的产生，这些动物就会失去长期记忆。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短时间内会依赖于海马体，之后它们就不再容易受到海马体的操纵。这个过程被称为“系统整合”，记忆可能通过这个过程从海马体转移到皮层。然而，最近的研究表明，在完成最初的细胞巩固过程后，记忆在被回忆时会再次参与海马体。这种现象现在被称为再巩固，它似乎依赖于蛋白质合成。这里出现了一个简单的问题，即记忆是否达到固定的稳定状态，或者它们是否在每次被激活时都会发生变化。当添加记忆“移动”的可能性时？一段时间后从大脑的一个区域转移到另一个区域，我们可能会对稳定记忆或其巩固的传统观点提出质疑。该领域的研究需要注意的是，大多数研究都使用茴香霉素等化学物质来抑制蛋白质合成。最近的研究表明，蛋白质合成抑制剂可诱导 mRNA 表达，这一过程称为“超诱导”。它可以通过三种方式之一发生，包括 (i) mRNA 稳定，(ii) 细胞内信号通路的激活，或 (iii) 干扰转录下调。此外，茴香霉素已被证明可以激活哺乳动物细胞中的 MAP 激酶途径。为了克服这些缺点，在细胞和系统水平上研究记忆巩固需要在活体动物中进行蛋白质合成敲低的诱导性遗传操作。 自去年以来，我们启动了一个项目，开发小鼠大脑中蛋白质合成的诱导性基因抑制。众所周知，双链 RNA 依赖性蛋白激酶 R (PKR) 会磷酸化 eIF2a，eIF2a 是蛋白质翻译过程中启动肽延伸的关键酶，并抑制细胞中大多数蛋白质的合成。利用给药后激酶激活所需的 PKR 结构域的诱导二聚化，姜志宏博士制备了在 Cam 激酶 II 启动子控制下的 loxP-LacZ-loxP-FKBP-PKR 的 cDNA 构建体。转基因核心设施（Jim Pickel 博士）将构建体注射到小鼠卵中以产生转基因小鼠，她已经获得了多个在小鼠前脑中显示出 LacZ 高表达的品系。一旦这些品系与特定的 Cre 重组酶转基因品系（例如 CA1-Cre 品系）杂交，我们期望以诱导方式创建 CA1 限制性遗传蛋白合成敲低小鼠。在另一个项目中，她还尝试使用 BAC 转基因技术创建海马 CA1 限制性 Cre 转基因系。 FKBP-PKR与CA1-Cre杂交的双条件转基因系对于海马依赖性记忆系统巩固的研究将非常有益。该项目的关键点在于，在给药后Cre重组酶高表达的脑细胞中，蛋白质合成抑制是否足以发生。测试这个问题的实验正在进行中。