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.

项目概要/摘要 肌萎缩侧索硬化症（ALS）是一种相对罕见的疾病，会导致运动神经元变性 1:50,000 人在美国。由于大约 50 个不同基因的突变与 ALS 相关，因此存在着令人畏惧的问题 治疗方法可能需要开发许多单独的疗法。因此，识别 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，目前很多努力都集中在路径上 与蛋白质停滞、自噬和细胞应激相关，因为蛋白质聚集是 ALS 的一个常见特征。 该提案采用了一种补充方法，通过检查基本电机所发挥的机械作用 受 ALS 影响的神经元特异性 microRNA (miR-218)。 用于识别散发性和家族性 ALS 中失调的 microRNA 的基因表达研究始终检测 miR-218 的下调。最近对 ALS 患者的一项分析发现，一群人的 miR-218 发生突变， 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 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，导致无法维持运动的基因失调模式 神经元。相反，预计异位 miR-218 可能会恢复运动神经元中正确的基因表达 并对抗 ALS。虽然之前的研究已经证实 miR-218 控制运动神经元连接 在胚胎中，这项资助的目标是确定成年运动神经元中由 miR-218 控制的基因网络 利用遗传学通过精确的时空控制来降低（目标 1）和升高（目标 2）miR-218。一个柔韧的- 创建了 miR-218 等位基因，并在成年运动神经元中通过 Cre 介导的缺失后发现 神经肌肉缺陷的出现——表明 miR-218 是成熟运动神经元中的关键调节分子。在 目标1 miR-218将在成年小鼠运动神经元和下一代RNA测序中被条件性删除 来自单核的 miR-218 将用于 (1a) 揭示成人运动神经元中的 miR-218 基因网络，以及 (1b) 交叉 将 miR-218 调节的基因与 ALS 小鼠模型中失调的基因相关联。在目标 2 中，miR-218 将 在小鼠中条件性（异位）表达，以 (2a) 定义 mir-218 的非细胞自主效应，以及 (2b) 建立运动神经元中 miR-218 基线水平的模型，该水平对于潜在的 ALS 疗法是可耐受的。 这些研究将为未来直接测试miR-218是否可用于减弱的实验铺平道路 肌萎缩侧索硬化症。为了使这成为可能，提交了独立但补充的 R03（参见补充 应用），让其他患有 ALS 模型的人能够使用我们的 miR-218 试剂探索这种有希望的可能性。这 R21 的资助是了解 miR-218 作用机制及其可能作用的重要一步 用于减弱 ALS。