MiR-218 regulatory networks in adult mice and its relationship to ALS

成年小鼠的 MiR-218 调控网络及其与 ALS 的关系

• 批准号: 10196817
• 负责人: SAMUEL L. PFAFF
• 金额: $ 52.91万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-15 至 2022-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10196817
• 关键词: ALS patients; Adult; Affect; Alleles; Amyotrophic Lateral Sclerosis; Astrocytes; Attenuated; Automobile Driving; Autophagocytosis; Behavioral; Birth; Cell Nucleus; Cells; Cellular Stress; Cessation of life; Communities; Complex; Data; Defect; Development; Disease; Distal; Down-Regulation; Embryo; Engineering; Environment; Extracellular Space; Future; Gene Expression; Gene Mutation; Genes; Genetic; Goals; Grant; Hand Strength; Health; Hindlimb; Homeostasis; Interneurons; Intervention; Light; Link; LoxP-flanked allele; Maintenance; Maps; Mediating; MicroRNAs; Modeling; Molecular; Motor; Motor Neuron Disease; Motor Neurons; Mus; Muscular Atrophy; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuromuscular Junction; Nodal; Pathway interactions; Pattern; Phenotype; Play; Proteins; RNA metabolism; Rare Diseases; Reagent; Research; Risk Factors; Role; Sampling; Specificity; Spinal; Spinal Muscular Atrophy; Synapses; Testing; Therapeutic; Therapeutic Agents; Time; Toxic effect; Work; amyotrophic lateral sclerosis therapy; cell type; cohort; design; disability; early embryonic stage; experimental study; familial amyotrophic lateral sclerosis; gene therapy; motor control; motor neuron degeneration; mouse model; neuromuscular; neuron loss; new therapeutic target; next generation; protein aggregation; side effect; spatiotemporal; success; therapeutic target; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT Amyotrophic lateral sclerosis (ALS) is a relatively-rare disease that leads to motor neuron degeneration in 1:50,000 people in the US. Since mutations in ~50 different genes have been linked to ALS, there is the daunting possibility that treatments will require the development of many separate therapies. Consequently, identification of the shared pathways and key nodal points affected in multiple forms of sporadic and familial ALS could provide greater impact for the overall ALS community. In this regard much effort is currently focused on pathways relevant to protein-stasis, autophagy and cell stress, since protein aggregates are a common feature of ALS. This proposal takes a complimentary approach by examining the mechanistic role played by an essential motor neuron-specific microRNA (miR-218) that is affected by ALS. Gene expression studies to identify microRNAs dysregulated in sporadic and familial ALS consistently detect downregulation of miR-218. A recent analysis of ALS patients found a cohort with mutations in miR-218, suggesting insufficient miR-218 levels/activity may be a risk factor for the disease. Because many ALS-linked genes affect RNA metabolism and microRNA processing complexes, it is hypothesized that miR-218 activity is downregulated in many types of ALS leading to a pattern of gene dysregulation that fails to sustain motor neurons. Conversely, it is predicted that ectopic miR-218 may restore proper gene expression in motor neurons and counteract ALS. While previous studies have established that miR-218 controls motor neuron connectivity in embryos, the goal of this grant is to identify the gene networks controlled by miR-218 in adult motor neurons using genetics to decrease (aim 1) and elevate (aim 2) miR-218 with precise spatiotemporal control. A floxed- miR-218 allele was created and following Cre-mediated deletion in adult motor neurons it was found that neuromuscular defects arose - indicating miR-218 is a critical regulatory molecule in mature motor neurons. In Aim 1 miR-218 will be conditionally deleted in adult mouse motor neurons and next generation RNA sequencing from single-nuclei will be used to (1a) uncover the miR-218 gene network in adult motor neurons, and (1b) cross- correlate miR-218-regulated genes with dysregulated genes in mouse models of ALS. In Aim 2, miR-218 will be conditionally (ectopically) expressed in mice to (2a) define non-cell-autonomous effects of mir-218, and (2b) model baseline levels of miR-218 in motor neurons that would be tolerable for potential ALS-therapies. These studies will pave the way for future experiments to directly test whether miR-218 can be used to attenuate ALS. To make this possible an independent but complementary R03 was submitted (see complimentary application) to allow others with ALS-models to explore this promising possibility with our miR-218 reagents. This R21 grant is an important step toward understanding the mechanism-of-action of miR-218 and how it might be used to attenuate ALS.

项目总结/摘要 肌萎缩侧索硬化症（ALS）是一种相对罕见的疾病，导致运动神经元变性， 1：50，000美国人由于约50种不同基因的突变与ALS有关， 治疗可能需要开发许多单独的疗法。因此，识别 在多种形式的散发性和家族性ALS中受影响的共享途径和关键节点可以提供 对整个ALS社区产生更大的影响。在这方面，目前的许多努力都集中在 这与蛋白质停滞、自噬和细胞应激有关，因为蛋白质聚集体是ALS的共同特征。 这一建议采取了一种补充的方法，通过检查一个重要的马达所发挥的机械作用， 神经元特异性microRNA（miR-218），受ALS影响。 基因表达研究，以确定在散发性和家族性ALS中失调的microRNA一致检测到 下调miR-218。最近对ALS患者的分析发现了一个miR-218突变的队列， 提示miR-218水平/活性不足可能是该疾病的危险因素。因为许多ALS相关的 基因影响RNA代谢和microRNA加工复合物，假设miR-218活性是 在许多类型的ALS中下调，导致基因失调的模式， 神经元相反，据预测，异位miR-218可以恢复运动神经元中的适当基因表达 并对抗肌萎缩侧索硬化症虽然先前的研究已经确定miR-218控制运动神经元连接， 在胚胎中，该基金的目标是确定成年运动神经元中由miR-218控制的基因网络 使用遗传学以精确的时空控制降低（目的1）和升高（目的2）miR-218。一个用牙线- 产生了miR-218等位基因，并在成年运动神经元中进行Cre介导的缺失后发现， 这表明miR-218是成熟运动神经元中的关键调节分子。在 目的1成年小鼠运动神经元中miR-218的条件性缺失及下一代RNA测序 将用于（1a）揭示成年运动神经元中的miR-218基因网络，以及（1b）交叉- 在ALS小鼠模型中将miR-218调节基因与失调基因相关联。在目标2中，miR-218将 在小鼠中条件性（异位）表达，以（2a）确定mir-218的非细胞自主作用，和（2b） 运动神经元中miR-218的模型基线水平对于潜在ALS疗法是可耐受的。 这些研究将为未来的实验铺平道路，直接测试miR-218是否可以用于减弱 人症为了使这一点成为可能，提交了一份独立但补充的R 03（见补充报告 应用），以允许其他ALS模型的人使用我们的miR-218试剂探索这种有希望的可能性。这 R21资助是理解miR-218作用机制及其可能机制的重要一步。 用于减轻肌萎缩侧索硬化症