MiR-193a-5p as a Regulator of Intersectin1-short and Clathrin-Mediated Endocytosis in Alzheimer's Disease.

项目摘要 阿尔茨海默氏病（AD）是痴呆的最常见形式，其特征在于发展为： 淀粉样蛋白β（Aβ）斑块和大脑中的神经元缠结。目前，AD的发病机制是 目前还不清楚，也没有可靠的工具来检测大脑的早期变化。Saugstad实验室的研究 揭示了一组在脑脊液（CSF）中减少的特定microRNA（miRNA）生物标志物， 其可以将AD患者与神经学正常对照分类。miRNAs调控转录后基因 通过与mRNA中的序列互补结合来表达，所述mRNA中的序列反过来调节翻译和表达 产生的蛋白质。然而，关于哪些mRNA是靶向的， 受AD CSF miRNAs调控的研究以及这些靶蛋白如何参与AD病理生理学。到 为了解决这一差距，我开发了一个生物信息学管道来预测AD CSF miRNAs的mRNA靶点， 将Intersectin 1-short（ITSN 1-s）鉴定为AD CSF miRNA miR-193 a-5 p靶向的蛋白质。免疫印迹 人死后海马的分析显示，与对照组相比， 控制然而，由于ITSN 1-s在多种脑细胞类型中表达，进一步的研究表明， 需要AD中ITSN 1-s表达增加的特定细胞类型来研究潜在机制 基础AD已发表的研究报告称，ITSN-s在星形胶质细胞和小胶质细胞中表达， ITSN 1-s的功能是减少网格蛋白介导的内吞作用（CME）。小胶质 已显示通过网格蛋白介导的机制摄取纤维状Aβ，AD中的CME破坏 与神经元中Aβ清除率降低有关。然而，目前尚不清楚小胶质细胞中的ITSN 1-s是否 有助于AD的病理生理学。该提案将评估miR-193 a-5 p对 ITSN 1-s及其在CME和AD中的功能意义为了做到这一点，我提出以下建议 目标：1.建立AD小胶质细胞和神经元中miR-193-a-5 p和ITSN 1-s的细胞表达; 2. 研究miR-193 a-5 p与ITSN 1-s mRNA的相互作用及其对ITSN 1-s蛋白的影响 体外和体内表达;和3.确定miR-193 a-5 p水平对AD中CME和Aβ摄取的作用 小胶质细胞最终，这些研究将更深入地了解ITSN 1-s对AD的监管以及如何监管 这可能有助于AD病理学。此外，这一框架可用于未来的研究，以评估 AD中循环miRNA生物标志物的生物学后果。

PROJECT SUMMARY Alzheimer’s disease (AD) is the most common form of dementia and is characterized by the development of amyloid beta (Aβ) plaques and neurofibrillary tangles in the brain. Currently, the pathogenesis underlying AD is unclear and there are no reliable tools to detect early changes in the brain. Studies in the Saugstad lab revealed a specific set of microRNA (miRNA) biomarkers that are decreased in cerebrospinal fluid (CSF), and which can classify AD patients from neurologically normal controls. MiRNAs regulate post-transcriptional gene expression via complementary binding to sequences in mRNA that in turn regulate translation and expression of the resulting protein. However, there is a knowledge gap regarding which mRNAs are targeted and regulated by the AD CSF miRNAs and how these target proteins may contribute to AD pathophysiology. To address this gap, I developed a bioinformatics pipeline to predict mRNA targets of the AD CSF miRNAs, and identified Intersectin1-short (ITSN1-s) as a protein targeted by the AD CSF miRNA, miR-193a-5p. Immunoblot analysis of human post mortem hippocampus showed significant increases in ITSN1-s protein in AD relative to control. However, as ITSN1-s is expressed in multiple brain cell types, further studies that determine the specific cell type(s) with increased ITSN1-s expression in AD are needed to examine potential mechanisms underlying AD. Published studies report that ITSN-s is expressed in astrocytes and microglia, with low expression in neurons, and that ITSN1-s functions to reduce clathrin-mediated endocytosis (CME). Microglia have been shown to uptake fibrillar Aβ through a clathrin-mediated mechanism, and CME disruption in AD has been linked to reduced Aβ clearance in neurons. However, it is not known whether ITSN1-s in microglia contributes to this pathophysiology in AD. This proposal will assess the regulatory role of miR-193a-5p on ITSN1-s and its functional significance in CME and AD. In order to accomplish this, I propose the following aims: 1. establish the cellular expression of miR-193-a-5p and ITSN1-s in AD microglia and neurons; 2. investigate the interaction between miR-193a-5p and ITSN1-s mRNA and the effects on ITSN1-protein expression in vitro and in vivo; and 3. determine the role of miR-193a-5p levels on CME and Aβ uptake in AD microglia. Ultimately, these studies will provide a deeper understanding of ITSN1-s regulation in AD and how this may contribute to AD pathology. In addition, this framework can be used in future studies to evaluate the biological consequences of circulating miRNA biomarkers in AD.