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: 9397913
• 负责人: Robert W Greene
• 金额: $ 63.79万
• 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/9397913
• 关键词: Animals; Anterior; Behavior; Behavioral; Cells; Circadian Rhythms; Contralateral; Dependence; Detection; Discipline; Employee Strikes; 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; Structure; Synapses; Technology; Testing; Time; Tissues; Transcript; Transcriptional Activation; Transfection; Vertebral column; base; brain health; calmodulin-dependent protein kinase II; 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/摘要众多研究始终显示出大量的变化 跨睡眠/唤醒状态的转录组。我们基于RNASEQ技术的初步证据表明 响应四个小时的睡眠，在恢复睡眠中差异表达了2800多个基因 剥夺与对照条件相比。这些DEG富含与突触功能有关的基因 对于转录因子的靶标，肌细胞增强因子2（MEF2C）控制着突触强度。 睡眠功能的领先提议提出了醒来过程中皮质突触强度的总体积累 经验和随后睡眠期间突触强度的降低。 MEF2主动形式的表达 降低突触强度。我们的初步证据表明，四个小时的睡眠剥夺增加了 MEF2（活动形式）与PMEF2（抑制形式）比率。这使我们提出了这样一个假设，即睡眠 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上的谷氨酸突触强度。 我们计划通过首先建立跨行为的差异转录组表达来检验这一假设 野生型小鼠中的状态条件，然后将其在这些相同条件的表达中进行比较 有条件的MEF2C敲除突变体（KO将仅限于谷氨酸前脑神经元）。 我们计划使用三个睡眠指标来表征MEF2C突变体中的睡眠需求/分辨率表型 需要。将在基线和睡眠剥夺（SD）条件下检查突变体和野生型对照。 我们的初步证据表明，稳态睡眠反应在突变体中丢失。 MEF2的磷酸化状态确定其活性，因此我们将评估MEF2的磷酸化状态 与行为状态条件相关。此外，由于高去甲肾上腺素能活性会引起裂解 HDAC4的N末端以抑制MEF2转录活性，我们将评估该N末端产品 在睡眠唤醒状态下也是如此。 我们将表征与睡眠有关的，依赖MEF2的突触的结构和功能变化，包括 从形态定义的脊柱数量，树突结构和MEPSC频率和振幅从 2-3层和5-6个锥体神经元，在额叶皮质叶（前扣带回）体外。 该提案将为跨睡眠/唤醒状态提供最早基于RNASEQ的分析之一 我们是研究人员有用的资源，以帮助调查和理解大型 响应长时间醒来而发生的转录组变化。它可以提供必不可少的开始 鉴定与睡眠相关的细胞自主信号级联和分子靶标的点， 对大脑健康很重要。