MicroRNAs in schizophrenia

项目总结 本修订后的R01申请是根据PAR-14-309提交的，旨在了解 微小RNA富含皮质小白蛋白表达(PV)GABA能中间神经元的机制 调节这些细胞的活动和它们控制下的行为。在前额叶皮质(PFC)，microRNA-206 (MIR-206)高度富含PV中间神经元，其在死后PFC中的表达水平与 精神分裂症和双相情感障碍患者的精神障碍。在令人兴奋的初步数据中，我们表明新产生的 MIR-206基因敲除小鼠表现出细胞和认知缺陷，这与以下假设一致 MicroRNA调节PFC中PV中间神经元的功能。这项提议的目标有三个：第一，蜂窝和 MiR-206 KO小鼠和表达miR-206的KO小鼠的行为异常 大脑皮层PV中间神经元的“获救”，将被评估。为了实现这一目标，我们生成了miR-206 在PV神经元中表达Cre重组酶的基因敲除小鼠。我们将使用依赖于Cre的表达载体 在皮质PV中间神经元中重新表达原本缺失的miRNA。然后我们将评估 MiR-206皮层PV中间神经元与相邻锥体神经元之间的突触联系 KO小鼠、恢复表达miR-206的KO小鼠和适当的对照组 组。我们还将描述大脑皮质PV中间神经元控制的认知和情绪的各个方面 在这些老鼠身上。第二，miR-206控制PV中间神经元活性的细胞内机制， 因此将研究mPFC函数。我们将使用高通量测序技术对由 用交联式免疫沉淀(HITS-CLIP)鉴定皮层PV中miR-206靶向基因 中间神经元。我们将使用3‘UTR荧光素酶报告来验证所识别的基因是miR-206的直接靶点 检测、RNA表达分析和蛋白质免疫印迹。第三，miR-1的分子机制。 206控制PV中间神经元的活动，因此mPFC功能将被研究。具体来说，我们将 利用在体CRISPR技术删除PV中间神经元miR-206靶向的最有前景的基因 并评估其行为后果。本申请中提出的尖端实验将 有助于更好地理解皮质PV中间神经元活动的分子机制 并可能对PV中间神经元相关精神障碍的病理生理学产生新的见解。

PROJECT SUMMARY This revised R01 application is submitted in response to PAR-14-309 and seeks to understand the mechanisms by which a microRNA enriched in cortical parvalbumin-expressing (PV) GABAergic interneurons regulates the activity of these cells and behaviors under their control. In prefrontal cortex (PFC), microRNA-206 (miR-206) is highly enriched in PV interneurons and its expression levels in post-mortem PFC correlate with psychosis in schizophrenia and bipolar patients. In exciting preliminary data, we show that newly generated miR-206 knockout mice demonstrate cellular and cognitive deficits consistent with the hypothesis that this microRNA regulates PV interneuron function in PFC. The goals of this proposal are threefold: First, cellular and behavioral abnormalities in miR-206 KO mice, and in KO mice in which miR-206 expression has been “rescued” in cortical PV interneurons, will be assessed. To accomplish this Aim we have generated miR-206 knockout mice that express Cre recombinase in PV neurons. We will use a Cre-dependent expression vector to re-express the otherwise deleted miRNA in cortical PV interneurons. We will then assess the strength of synaptic connectivity between cortical PV interneurons and neighboring pyramidal neurons in PFC of miR-206 KO mice, KO mice with rescued miR-206 expression in cortical PV interneurons, and appropriate control groups. We will also characterize aspects of cognition and emotionality controlled by cortical PV interneurons in these mice. Second, the intracellular mechanisms by which miR-206 controls activity of PV interneurons, and hence mPFC function, will be investigated. We will use High-Throughput Sequencing of RNA isolated by CrossLinking ImmunoPrecipitation (HITS-CLIP) to identify genes targeted by miR-206 in cortical PV interneurons. We will verify that identified genes are direct targets for miR-206 using 3'UTR luciferase reporter assays, RNA expression analysis and protein immunoblotting. Third, the molecular mechanisms by which miR- 206 controls activity of PV interneurons, and hence mPFC function, will be investigated. Specifically, we will use in vivo CRISPR technology to delete the most promising genes targeted by miR-206 in PV interneurons and assess the behavioral consequences. The cutting-edge experiments proposed in this application will facilitate greater understanding of the molecular mechanisms by which cortical PV interneuron activity is regulated and may yield new insights into the pathophysiology of PV interneuron-related psychiatric disorders.