The Impact of Sleep Deprivation on Translation

描述（由申请人提供）：睡眠不足会导致海马突触可塑性和海马依赖性记忆储存的缺陷。然而，导致睡眠不足的分子和细胞机制尚不清楚。我们最近的工作表明，睡眠剥夺会改变蛋白质合成，减少翻译起始。在这个应用中，提出了两个特定的目标，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 （4EBP2）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。调控蛋白翻译的一种途径是哺乳动物雷帕霉素复合物1 （mTORC1）信号通路，它通过S6激酶和4EBP2两条下游途径起作用。我们发表的工作和初步研究表明，睡眠剥夺会减弱mTORC1信号，导致4EBP2的总磷酸化水平降低。然而，尚不清楚这些睡眠剥夺引起的4EBP2表达变化是否对海马体有功能影响。在Specific Aim 1中，基因技术将用于确定4EBP2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

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.