Sleep and the Functional Genomics of Synaptic Modulation

睡眠与突触调节的功能基因组学

• 批准号: 9900054
• 负责人: Robert W Greene
• 金额: $ 61.8万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9900054
• 关键词: Animals; Anterior; Behavior; Behavioral; Cells; Contralateral; Dependence; Detection; Discipline; Foundations; Frequencies; Genes; Genetic Transcription; Glutamates; HDAC4 gene; Harvest; Hour; In Vitro; Investigation; Knock-out; Knockout Mice; Lead; Lobe; LoxP-flanked allele; Mental disorders; Messenger RNA; Metabolic syndrome; Molecular; Molecular Target; Morphology; Mus; Muscle Cells; N-terminal; Nerve Degeneration; Neurobiology; Neurons; Pathologic; Pathology; Pathway interactions; Phenotype; Phospho-Specific Antibodies; Phosphorylation; Prosencephalon; Pyramidal Cells; Recovery; Research Personnel; Resolution; Resources; Role; Siblings; Signal Transduction; Sleep; Sleep Deprivation; Sleep disturbances; Structure; Synapses; Technology; Testing; Time; Tissues; Transcript; Transcriptional Activation; Transfection; Vertebral column; Wild Type Mouse; base; brain health; calmodulin-dependent protein kinase II; circadian; conditional knockout; differential expression; enhancing factor; evidence base; experience; frontal lobe; functional genomics; hippocampal pyramidal neuron; indexing; mutant; neurobiological mechanism; noradrenergic; recombinase-mediated cassette exchange; response; stem; synaptic function; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

7. Project Summary/Abstract Numerous studies have consistently shown a remarkably large change in the transcriptome across sleep/wake states. Our preliminary evidence based on RNAseq technology suggests more than 2800 genes are differentially expressed in recovery sleep in response to four hours of sleep deprivation, compared to control conditions. These DEG's are enriched for genes related to synaptic function and for targets of a transcription factor, myocyte enhancing factor 2 (MEF2C), that controls synaptic strength. A leading proposal for the function of sleep posits an overall buildup of cortical synaptic strength during waking experience and a decrease of synaptic strength during ensuing sleep. Expression of the active form of Mef2 decreases synaptic strength. Our preliminary evidence suggests that four hours of sleep deprivation increases the MEF2 (the active form) to pMEF2 (the repressive form) ratio. This has lead us to the hypothesis that sleep- need modulates the activity of the transcription factor, MEF2C, to alter the expression of downstream mRNA to reduce glutamate synapse strength on forebrain cortical glutamate neurons. We plan to test this hypothesis by first establishing the differential transcriptome expression across behavioral state conditions in wildtype mice and then to compare it across these same conditions to the expression in conditional Mef2c knockout mutants (the ko will be restricted to glutamate forebrain neurons). We plan to characterize a sleep need/resolution phenotype in the Mef2c mutant using three indices of sleep need. Mutants and wildtype controls will be examined under baseline and sleep deprivation (SD) conditions. Our preliminary evidence shows that the homeostatic sleep response is lost in the mutants. The phosphorylation state of MEF2 determines its activity so we will assess the phosphorylation state of MEF2 in correlation with behavioral state condition. Additionally, since high noradrenergic activity can cause cleavage of the N-terminal of HDAC4 to inhibit MEF2 transcriptional activity, we will assess this N-terminal product across sleep wake states as well. We will characterize sleep-related, Mef2 dependent structural and functional changes of synapses, including morphologically defined spine number, dendritic structure and mEPSC frequency and amplitude recorded from layer 2-3 & 5-6 pyramidal neurons, in frontal cortical lobe (the anterior cingulate) in vitro. This proposal will provide one of the first comprehensive RNAseq based analyses across sleep/wake states that we be a useful resource for investigators to aid in the investigation and understanding of the large transcriptomic change that takes place in response to prolonged waking. It can provide an essential starting point for the identification of sleep related cell autonomous signaling cascades and molecular targets, important to brain health.

7.项目摘要/摘要许多研究一直表明，在 睡眠/清醒状态的转录组。我们基于RNAseq技术的初步证据表明 超过2800个基因在恢复睡眠中对四个小时的睡眠做出反应 剥夺，与对照条件相比。这些DEG富含与突触功能相关的基因 对于控制突触强度的转录因子--肌细胞增强因子2(MEF2C)的靶标。 一项关于睡眠功能的主要提议提出，清醒时皮质突触强度总体增强。 在随后的睡眠中经历和突触强度的下降。MEF2活性形式的表达 降低突触强度。我们的初步证据表明，睡眠不足四个小时会增加 MEF2(主动形式)与pMEF2(抑制形式)的比率。这让我们得出这样的假设：睡眠-- 需要调节转录因子MEF2C的活性，以改变下游mRNA的表达 降低前脑皮质谷氨酸神经元的谷氨酸突触强度。 我们计划通过首先建立不同行为的不同转录组表达来检验这一假说 描述野生型小鼠的情况，然后将其在这些相同的条件下与 条件性MEF2C基因敲除突变体(KO将仅限于谷氨酸前脑神经元)。 我们计划使用三种睡眠指数来表征MEF2C突变体中的睡眠需要/解决表型 需要。突变型和野生型对照将在基线和睡眠剥夺(SD)条件下进行检查。 我们的初步证据表明，在突变体中丢失了动态平衡睡眠反应。 MEF2的磷酸化状态决定了其活性，因此我们将评估MEF2的磷酸化状态 与行为状态状况相关。此外，由于高去甲肾上腺素能活动会导致卵裂 为了抑制MEF2的转录活性，我们将评估HDAC4的N端产物 在睡眠清醒状态下也是如此。 我们将描述与睡眠相关的、依赖MEF2的突触的结构和功能变化，包括 从形态上定义的棘数、树突结构以及mEPSC的频率和幅度 2-3层和5-6层锥体神经元，在体外额叶皮质(前扣带回)。 该提案将提供首批基于RNAseq的睡眠/清醒状态综合分析之一 我们是调查人员的有用资源，以帮助调查和了解 由于长时间醒着而发生的转录改变。它可以提供一个重要的起点 识别睡眠相关细胞自主信号级联和分子靶标的点， 对大脑健康很重要。