Cell type specific analysis of miRNA expression

摘要 许多神经障碍的特征在于特异性神经功能障碍和/或丧失。 神经元细胞群体和这种细胞类型特异性脆弱性的机制仍然很差， 明白有人提出，在这些疾病中观察到的差异性神经元脆弱性产生 细胞类型特异性基因表达调控程序的破坏， 受影响神经元的生理特性和功能。microRNAs（miRNAs）是一种小的非编码RNA 在转录后水平参与基因表达网络的调控。研究，包括我们的， 在几种神经系统疾病的致病性中涉及改变的miRNA表达。这里我们 建议开发一种新的分子方法来研究神经细胞类型特异性 miRNA功能的改变在神经系统疾病的致病性中发挥作用。该方法依赖于 基于重组腺相关病毒（rAAV）的Cre重组酶依赖性遗传Flp切除开关 (FLEX开关），其限制异位递送的miRNA结合蛋白Argonaute-2的表达， Cre表达细胞。我们称这种方法为miFLAGO（miRNA相关，FLEX开关调节，AGO 2）。 Aim-1中的实验将验证miFLEX在成人小脑中的功能性和再现性。 在浦肯野细胞中选择性表达Cre重组酶的转基因小鼠。浦肯野细胞特异性 miFAGO-miRNA复合物将被分离并用于构建高通量RNA的miRNA文库。 测序分析在Aim-2中，我们将使用miFLAGO来研究细胞类型特异性变化在 miRNA在脊髓小脑共济失调1型小鼠模型致病性中的作用，一种多聚谷氨酰胺 由ATXN 1基因中CAG重复扩增引起的疾病。该提案的短期目标是 为研究界提供了一个有效的新的分子工具，用于研究细胞类型特异性 miRNA功能从长远来看，我们的目标是微调我们对神经细胞类型特异性基因表达的理解 监管程序以及它们如何介导神经系统疾病的差异脆弱性， 发现新的治疗途径

Abstract A number of neurological disorders are characterized by the preferential dysfunction and/or loss of specific neuronal cell populations and the mechanisms underlying this cell type specific vulnerability remain poorly understood. It has been proposed that the differential neuronal vulnerability observed in these disorders arises from disruptions in cell type specific gene expression regulatory programs responsible for maintaining the physiological identity and function of the affected neurons. MicroRNAs (miRNAs) are small, non-coding RNAs involved in the regulation of gene expression networks at the posttranscriptional level. Studies, including ours, have implicated altered miRNA expression in the pathogenicity of several neurological disorders. Here, we propose the development of a novel molecular approach to investigate the role that neural cell type specific changes in miRNA function play in the pathogenicity of neurological diseases. The approach relies on a recombinant adeno-associated virus (rAAV)-based, Cre recombinase-dependent genetic Flp-excision switch (FLEX switch) that restricts the expression of an ectopically delivered miRNA-binding protein Argonaute-2 to Cre expressing cells. We call this approach miFLAGO (miRNA-associated, FLEX switch regulated, AGO2). Experiments in Aim-1 will validate the functionality and reproducibility of miFLEX in the cerebellum of adult transgenic mice engineered to selectively express Cre recombinase in Purkinje cells. Purkinje cell-specific miFAGO-miRNA complexes will be isolated and used to build miRNA libraries for high-throughput RNA sequencing analysis. In Aim-2, we will use miFLAGO to investigate the role that cell type specific changes in miRNA function play in the pathogenicity of a mouse model of Spinocerebellar ataxia type-1, a polyglutamine disorder caused by the expansion of a CAG repeat in the ATXN1 gene. The short-term goal of this proposal is to provide the research community with a validated novel molecular tool for the study of cell type specific miRNA function. Long-term, we aim to fine-tune our understanding of neural cell type specific gene expression regulatory programs and how they might mediate differential vulnerability in neurological disease, with the goal of identifying novel therapeutic routes.