The Impact of Sleep Deprivation on Translation

描述（由申请人提供）：睡眠丧失导致海马突触可塑性和海马依赖性记忆储存缺陷。然而，睡眠剥夺的这些影响背后的分子和细胞机制仍不清楚。我们最近的研究表明，睡眠剥夺会改变蛋白质合成，减少翻译起始。在本申请中，提出了两个特定目的，即使用新的遗传方法来调节海马真核起始因子结合蛋白2（4 EBP 2）的表达，以研究睡眠剥夺如何影响翻译，并确定影响认知的分子后果。调节蛋白质翻译的一种途径是哺乳动物雷帕霉素复合物靶蛋白1（mTORC 1）信号传导，其通过两个下游途径S6激酶和4 EBP 2起作用。我们发表的工作和初步研究表明，睡眠剥夺减弱mTORC 1信号传导，导致4 EBP 2的总水平和磷酸化水平降低。然而，目前尚不清楚这些睡眠剥夺诱导的4 EBP 2表达的变化是否在海马中具有功能性影响。在具体目标1中，将使用遗传技术来确定4 EBP 2和海马蛋白翻译起始在睡眠剥夺后观察到的记忆和可塑性缺陷中的作用。翻译起始的机制 在具体目标1中将研究睡眠剥夺对翻译的影响，但翻译受损的分子后果尚未得到表征。因此，在特定目标2A中，我们将使用新的遗传工具，利用最近开发的RNA标签和相关蛋白（TRAP）技术的恢复，选择性地分离核糖体结合的mRNA从海马兴奋性锥体神经元。我们将采用下一代测序技术来鉴定在睡眠剥夺期间进行主动翻译的信使RNA，以确定睡眠剥夺引起的分子变化。在Specific Aim 2B中，这些新鉴定的靶向mRNA将在Specific Aim 1的4 EBP 2-AAV小鼠中进行验证，以确定哪种特定蛋白质表达恢复了记忆和翻译。睡眠剥夺是我们社会中一个重要的公共卫生问题，数百万人睡眠不足，极大地影响了生活质量。此外，许多患有精神和神经退行性疾病的人，包括抑郁症、精神分裂症和阿尔茨海默氏病，也会遭受睡眠不足的困扰。该提案旨在扭转睡眠剥夺对翻译的负面影响及其随后的认知障碍。与睡眠不足相关的细胞机制和分子后果的知识有望导致新的治疗方法的发展，以减轻睡眠剥夺导致的认知缺陷。

DESCRIPTION (provided by applicant): Sleep loss produces deficits in hippocampal synaptic plasticity and hippocampus-dependent memory storage. However, the molecular and cellular mechanisms that underlie these effects of sleep deprivation remain unclear. Our recent work suggests that sleep deprivation alters protein synthesis, reducing translation initiation. In this application, two Specific Aims are proposed that use novel genetic approaches to modulate hippocampal eukaryotic initiation factor-binding protein 2 (4EBP2) expression to study how sleep deprivation affects translation and to identify the molecular consequences that impact cognition. One pathway that regulates protein translation is mammalian target of rapamycin complex 1 (mTORC1) signaling, which acts through two downstream pathways, S6 kinase and 4EBP2. Our published work and preliminary studies show that sleep deprivation attenuates mTORC1 signaling leading to reduced total and phosphorylated levels of 4EBP2. However, it is not known whether these sleep deprivation-induced changes in 4EBP2 expression have functional effects in the hippocampus. In Specific Aim 1, genetic techniques will be used to determine the role of 4EBP2 and hippocampal protein translation initiation in the memory and plasticity deficits observed after sleep deprivation. The mechanism by which translation initiation is impaired with sleep deprivation will be investigated in Specific Aim 1, but the molecular consequences of impaired translation has yet to be characterized. Therefore, in Specific Aim 2A we will use novel genetic tools that utilize recently developed RNA tagging and recovery of associated proteins (TRAP) technology to selectively isolate ribosome-bound mRNA from excitatory pyramidal neurons of the hippocampus. We will employ next generation sequencing to identify the messenger RNAs that are undergoing active translation during sleep deprivation to define the molecular changes caused by sleep deprivation. In Specific Aim 2B, these newly identified targeted mRNAs will be validated in the 4EBP2-AAV mice from Specific Aim 1 to determine which specific protein expression is restored with rescued memory and translation. Sleep deprivation is a significant public health issue in our society with millions of people obtaining insufficient sleep that greatly affects quality of life. Additionally, many people who hae psychiatric and neurodegenerative disorders, including depression, schizophrenia, and Alzheimer's disease, also suffer from sleep loss. This proposal seeks to reverse the negative impact of sleep deprivation on translation and its subsequent impairments in cognition. Knowledge of the cellular mechanism and molecular consequences associated with sleep loss promises to lead to the development of novel therapeutic approaches to alleviate the cognitive deficits that result from sleep deprivation.