MicroRNAs in schizophrenia

项目摘要 此修订的R 01申请是为了响应PAR-14-309而提交的，旨在了解 表达小白蛋白的皮质GABA能中间神经元中富含microRNA的机制 调节这些细胞的活动和它们控制下的行为。在前额叶皮层（PFC），microRNA-206 （miR-206）在PV中间神经元中高度富集，其在死后PFC中的表达水平与 精神分裂症和躁郁症患者的精神病。在令人兴奋的初步数据，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miR-206基因敲除小鼠的细胞和认知缺陷的假设一致。 microRNA调节PFC中PV中间神经元的功能。 在miR-206 KO小鼠中，以及在其中miR-206表达已被抑制的KO小鼠中， 将评估皮质PV中间神经元中的“拯救”。为了实现这一目标，我们产生了miR-206 在PV神经元中表达Cre重组酶的敲除小鼠。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在PFC中皮质PV中间神经元和相邻锥体神经元之间的突触连接 KO小鼠、在皮质PV中间神经元中具有拯救的miR-206表达的KO小鼠和适当的对照 组我们还将描述由皮层PV中间神经元控制的认知和情感方面的特征 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功能。我们将使用高通量测序的RNA分离， 用交联免疫沉淀法（HITS-CLIP）鉴定皮质PV中miR-206靶向的基因 中间神经元我们将使用3 'UTR荧光素酶报告基因验证所鉴定的基因是miR-206的直接靶点 分析、RNA表达分析和蛋白质免疫印迹。第三，miR-16的分子机制。 206控制PV中间神经元的活性，因此将研究mPFC功能。具体来说，我们将 使用体内CRISPR技术删除PV中间神经元中miR-206靶向的最有希望的基因 并评估其行为后果这项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， 调节，并可能产生新的见解PV神经元间相关的精神疾病的病理生理学。

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.