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: 10160964
• 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/10160964
• 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. 项目总结/摘要 许多研究一致表明， 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 超过 2800 个基因在恢复睡眠中因四小时睡眠而出现差异表达 与控制条件相比，剥夺。这些 DEG 富含与突触功能相关的基因 以及控制突触强度的转录因子肌细胞增强因子 2 (MEF2C) 的靶标。 关于睡眠功能的一项主要提议提出，清醒期间皮质突触强度的整体增强 随后睡眠期间的体验和突触强度下降。 Mef2活性形式的表达 降低突触强度。我们的初步证据表明，四个小时的睡眠不足会增加 MEF2（活性形式）与 pMEF2（抑制形式）的比率。这使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跨行为的差异转录组表达来检验这一假设。 状态野生型小鼠的条件，然后将其在这些相同条件下的表达与 条件性 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数来表征 Mef2c 突变体的睡眠需求/解决表型 需要。突变体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，突变体失去了稳态睡眠反应。 MEF2 的磷酸化状态决定其活性，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导致裂解 HDAC4 N 端抑制 MEF2 转录活性，我们将评估该 N 端产物 也跨越睡眠唤醒状态。 我们将描述与睡眠相关、Mef2 依赖性的突触结构和功能变化，包括 形态学定义的树突棘数量、树突结构以及记录的 mEPSC 频率和幅度 体外额皮质叶（前扣带回）2-3 层和 5-6 层锥体神经元。 该提案将提供第一个基于 RNAseq 的跨睡眠/唤醒状态的综合分析之一 我们成为调查人员的有用资源，帮助他们调查和了解大型案件 因长时间清醒而发生的转录组变化。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。

项目成果

Structure of cortical network activity across natural wake and sleep states in mice.

小鼠自然觉醒和睡眠状态下的皮质网络活动结构。

• 发表时间: 2020
• 影响因子: 3.7
• 作者: Ohyama, Kaoru;Kanda, Takeshi;Miyazaki, Takehiro;Tsujino, Natsuko;Ishii, Ryo;Ishikawa, Yukiko;Muramoto, Hiroki;Grenier, Francois;Makino, Yuichi;McHugh, Thomas J;Yanagisawa, Masashi;Greene, Robert W;Vogt, Kaspar E
• 通讯作者: Vogt, Kaspar E

Defining the Role of Interneuron N-Methyl-D-Aspartate Receptors in Prefrontal Cortex Inhibition.

定义中间神经元 N-甲基-D-天冬氨酸受体在前额皮质抑制中的作用。

• 发表时间: 2018-09-15
• 影响因子: 10.6
• 作者: Greene; Robert W
• 通讯作者: Robert W