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

项目总结 阿尔茨海默病(AD)是最常见的痴呆症形式，其特征是 大脑中的淀粉样β蛋白(Aβ)斑块和神经原纤维缠结。目前，AD的发病机制是 目前尚不清楚，也没有可靠的工具来检测大脑的早期变化。索格斯塔德实验室的研究 揭示了一组特定的microRNA(MiRNA)生物标记物，这些标记物在脑脊液(CSF)中减少 这可以将AD患者与神经学正常对照区分开来。MiRNAs调控转录后基因 通过与mRNA中的序列互补结合来表达，进而调节翻译和表达 所产生的蛋白质。然而，关于哪些mRNAs是靶向的，以及 由AD-CSF miRNAs调节，以及这些靶蛋白如何在AD病理生理学中起作用。至 为了解决这个问题，我开发了一条生物信息学管道来预测AD-CSF miRNAs的mRNA靶标，并且 Intersectin1-Short(ITSN1-S)是AD-CSFmiRNA(miR-193a-5p)的靶向蛋白。免疫印迹 对人死后海马区的分析表明，阿尔茨海默病患者ITSN1-S蛋白的表达显著高于对照组 控制力。然而，由于ITSN1-S在多种脑细胞类型中表达，进一步的研究确定了 需要特定的细胞类型(S)和ITSN1-S在AD中的表达增加来研究可能的机制 潜在的AD。已发表的研究报道，ITSN-S在星形胶质细胞和小胶质细胞中表达，低表达 研究结果表明，ITSN1-S基因在神经元中的表达具有抑制细胞骨架蛋白介导的内吞作用。小胶质细胞 已被证明通过网状蛋白介导的机制摄取纤维Aβ，而cME在AD中的破坏 与神经元中Aβ清除减少有关。然而，目前尚不清楚ITSN1-S是否存在于小胶质细胞中 对AD的这种病理生理学有贡献。该提案将评估miR-193a-5p对 ITSN1-S及其在继续医学教育和AD中的作用意义。为了实现这一目标，我提出以下建议 目的：1.建立miR-193-a-5p和ITSN1-S在AD小胶质细胞和神经元中的细胞表达； 研究miR-193a-5p与ITSN1-S的相互作用及其对ITSN1-蛋白的影响 3.确定miR-193a-5p水平对AD患者cME和Aβ摄取的影响。 小胶质细胞。最终，这些研究将对ITSN1-S在AD中的调控以及如何调控提供更深入的了解 这可能与AD的病理机制有关。此外，该框架可用于未来的研究，以评估 循环miRNA生物标记物在AD中的生物学后果。

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.