Tools for regulated expression control of miR-218

用于调控 miR-218 表达控制的工具

基本信息

批准号：
10196829
负责人：
SAMUEL L. PFAFF
金额：
$ 19.24万
依托单位：
SALK INSTITUTE FOR BIOLOGICAL STUDIES
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-05-15 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10196829
关键词：
ALS pathology ALS patients Adult Affect Alleles Amyotrophic Lateral Sclerosis Animal Model Applied Research Attenuated Basic Science Benchmarking Biological Assay Biology Birth C9ORF72 Cell Line Cells Communities Complex DNA Databases Data Defect Deposition Disease Distal Down-Regulation ES Cell Line Embryo Embryonic Development Enterobacteria phage P1 Cre recombinase Evolution Functional disorder Gene Expression Generations Genes Genetic Genotype Glioblastoma Glioma Goals Grant Hand Strength Health Hindlimb Human In Vitro Investigation Karyotype Laboratories Life Link LoxP-flanked allele Malignant neoplasm of central nervous system MicroRNAs Mining Modeling Molecular Motor Motor Neurons Mouse Strains Mus Muscle Mutant Strains Mice Mutation Nerve Degeneration Neurodegenerative Disorders Neuromuscular Junction Neurons Pancreas Pathology Physiology Play Prognosis Protocols documentation PubMed Publications RNA metabolism Reagent Research Research Personnel Resources Risk Factors Role Series Signal Transduction Spinal Spinal Cord Spinal Muscular Atrophy Synapses Tamoxifen Testing The Jackson Laboratory Therapeutic Therapeutic Agents Transgenes Transgenic Mice Validation Work amyotrophic lateral sclerosis therapy cell type cohort embryonic stem cell familial amyotrophic lateral sclerosis fetal in vitro testing in vivo interest medulloblastoma neuromuscular novel therapeutics pathogen pluripotency programs protein TDP-43 repository spatiotemporal therapeutic target tool

项目摘要

MiR-218 is a motor neuron-specific microRNA conserved across vertebrate evolution that is expressed by both fetal and adult lower motor neurons in humans and mice. There are two alleles of miR-218 in humans and mice, and null alleles of both miR-218-1 and miR-218-2 have been generated. It was found that embryos lacking both alleles of miR-218 develop normally with proper motor neuron numbers, however during the final week of embryonic development their motor neurons degenerate with a pathophysiology that mimics spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Although motor degeneration in ALS occurs in adults rather than at fetal stages as observed in miR-218 mutant mice, several findings provide further indirect support for a connection between ALS and miR-218: (1) Gene expression studies to identify microRNAs dysregulated in sporadic and familial ALS repeatedly detect downregulation of miR-218. (2) A recent analysis of ALS patients found a cohort with mutations in miR-218, suggesting insufficient miR-218 levels or activity may be a risk factor for the disease. (3) Many ALS-linked genes affect RNA metabolism and microRNA processing complexes, including TDP43, FUS and C9orf72, suggesting a plausible mechanism for miR-218 downregulation in ALS. (4) In preliminary studies a floxed-mutation of miR-218 was generated and it was found that deletion in adults leads to neuromuscular pathology. These observations suggest several obvious research possibilities, including tests to: (1) determine whether genetically-lowering miR-218 accelerates ALS-pathology in animal models and (2) whether ectopic miR-218 can reprogram the normal genetic circuits in motor neurons and attenuate ALS degeneration. The goal of this grant is to generate and provide miR-218 reagents to the ALS research community to accelerate the investigation of this promising new candidate for ALS-therapy. In Aim 1, the concentration levels of miR-218 will be defined in an allelic series of mouse lines. It is anticipated that these lines can be used by others to cross to their ALS-models systematically to raise the lower miR-218 levels in vivo. In Aim 2 mouse embryonic stem cell lines (mESC) will be derived from our allelic series of miR-218 lines which is anticipated to have value in culture assays since motor neurons with different miR-218 levels can easily and efficiently be generated from mES lines for investigating the cellular/molecular features of ALS in vitro. The reagents generated in this grant will be made widely available to researchers by depositing our mouse strains in the Jackson Laboratories repository and by maintaining a dedicated stock of mES cell lines for distribution. non- overlapping complementary R21 grant has also been submitted to characterize the genetic networks regulated by miR-218, in order to define mechanistically the motor neuron synaptic and survival modules regulated by this microRNA.

MiR-218 是一种在脊椎动物进化过程中保守的运动神经元特异性 microRNA，由人类和小鼠的胎儿和成人下运动神经元。人类和小鼠中的 miR-218 有两个等位基因，并生成了 miR-218-1 和 miR-218-2 的无效等位基因。研究发现胚胎同时缺乏这两种物质 miR-218 的等位基因正常发育，具有适当的运动神经元数量，但是在胚胎发育时，它们的运动神经元会退化，其病理生理学类似于脊髓肌肉萎缩症（SMA）和肌萎缩侧索硬化症（ALS）。尽管 ALS 运动退化发生在成人身上与在 miR-218 突变小鼠中观察到的胎儿阶段不同，一些发现提供了进一步的间接支持 ALS 和 miR-218 之间的联系：(1) 基因表达研究，以确定在散发性和家族性 ALS 反复检测到 miR-218 的下调。 (2) ALS患者近期分析发现了一个 miR-218 突变的队列，表明 miR-218 水平或活性不足可能是一个危险因素对于这种疾病。 (3) 许多 ALS 相关基因影响 RNA 代谢和 microRNA 加工复合物，包括 TDP43、FUS 和 C9orf72，表明 ALS 中 miR-218 下调的可能机制。 (4) 在初步研究中，产生了 miR-218 的 floxed 突变，并发现成人中的缺失会导致神经肌肉病理学。这些观察结果表明了几种明显的研究可能性，包括测试目的：(1) 确定基因降低的 miR-218 是否会加速动物模型中的 ALS 病理；(2) 异位 miR-218 是否可以重新编程运动神经元中的正常遗传回路并减弱 ALS 退化。这笔赠款的目标是为 ALS 研究界生成并提供 miR-218 试剂加速对这种有前途的 ALS 治疗新候选药物的研究。在目标 1 中，浓度 miR-218 的水平将在一系列等位基因小鼠品系中定义。预计这些线路可以使用其他人系统地交叉到他们的 ALS 模型，以提高体内较低的 miR-218 水平。瞄准 2 鼠标胚胎干细胞系 (mESC) 将源自我们的 miR-218 系等位基因系列，预计在培养测定中具有价值，因为具有不同 miR-218 水平的运动神经元可以轻松有效地由 mES 系生成，用于体外研究 ALS 的细胞/分子特征。试剂这笔赠款中产生的成果将通过将我们的小鼠品系存放在 Jackson Laboratories 储存库并维护 mES 细胞系的专用库存以供分发。非重叠互补的 R21 拨款也已提交，以描述受监管的遗传网络的特征通过 miR-218，以机械地定义受其调节的运动神经元突触和生存模块微小RNA。