METTL3-mediated regulation of motor neuron function

METTL3介导的运动神经元功能调节

• 批准号: 10781077
• 负责人: Francesco Lotti
• 金额: $ 41.13万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10781077
• 关键词: Address; Affect; Alzheimer' s Disease; Attention; Behavioral; Binding; Brain Stem; Cells; Cessation of life; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Cytoplasm; Data; Development; Disease; Ensure; Event; Failure; Foundations; Gene Expression; Gene Mutation; Genes; Genetic; Homeostasis; Impairment; Informal Social Control; Innovative Therapy; Light; Link; Liquid substance; Mediating; Membrane; Messenger RNA; Modification; Molecular; Morphology; Motor Neurons; Mus; Muscle; Muscle Fibers; Mutation; Nerve Degeneration; Nervous System; Neurites; Neurodegenerative Disorders; Neurons; Organelles; Output; Paralysed; Pathogenesis; Pathology; Pattern; Persons; Phase; Phenotype; Physiological; Post-Transcriptional Regulation; Predisposition; Process; Proteins; Public Health; RNA; RNA Processing; RNA Splicing; RNA-Binding Proteins; Regulation; Research; Role; Site; Spinal; Spinal Cord; Stress; TDP-43 aggregation; Techniques; Testing; Therapeutic; Transcript; Up-Regulation; Vertebral column; Work; age related; age related neurodegeneration; cost; demented; embryonic stem cell; epitranscriptome; epitranscriptomics; frontotemporal lobar dementia amyotrophic lateral sclerosis; human disease; innovation; insight; motor neuron degeneration; motor neuron function; mouse model; nerve supply; nervous system disorder; neuron loss; neuronal survival; neurotoxicity; novel; pharmacologic; posttranscriptional; protein TDP-43; protein expression; proteostasis; single nucleus RNA-sequencing; spatiotemporal; stem; therapeutically effective

SUMMARY Dementing neurodegenerative diseases affect millions of people and have an annual cost exceeding $380 billion dollars in the USA alone. To gain insights into potential effective therapeutic strategies for these incurable disorders, herein we focused our attention on the RNA-binding protein TAR DNA-binding protein-43 (TDP-43), whose altered subcellular distribution and role are thought to contribute to a number of dementing disorders including Alzheimer’s disease (AD) and the clinical spectrum of amyotrophic lateral sclerosis-frontotemporal dementia (ALS/FTD). TDP-43, like other RNA-binding proteins with low-complexity domains are involved in the formation of membrane-less organelles associated with RNA processing via liquid-liquid phase separation (LLPS) assembly. While disease-associated mutations in TDP-43 can disrupt LLPS and its ensuing roles, how wild-type TDP-43 subcellular distribution and function are altered in the majorly of AD and ALS/FTD cases, which are gene mutation negative, remain enigmatic. We propose, herein, to investigate the role of post-transcriptional regulatory mechanisms of TDP-43 mediated by N6-methyladenosine (m6A). This idea stems from our finding that TARDBP, the gene encoding for TDP-43, is among the m6A targets in embryonic stem cell-derived motor neurons (ES-MNs) and that its expression is under m6A epitranscriptomic regulation. Our central hypothesis is that loss of m6A marks on TDP-43 causes changes in its expression and in its subcellular distribution and role, which, in turn, may contribute to neurodegeneration. The rationale for this research is that, once it is known how m6A-mediated regulation of TDP-43 contributes to neurodegeneration, innovative genetic or pharmacological strategies can be devised for the treatment of these incurable human diseases. We propose to first (AIM 1) characterize the spatiotemporal susceptibility of MNs to m6A loss. Accordingly, we will define the pattern of MN loss in both spinal cord and brainstem in mice with Mettl3 depletion at both presymptomatic and symptomatic stages using a battery of behavioral, physiological and morphological techniques. Then, since our preliminary data show that TARDBP levels are increased when m6A is lost, in AIM 2, we will determine how the loss of m6A impairs TDP-43 expression and its subcellular localization. Our preliminary data also show that m6A depletion in ES-MNs results in reduced survival and neurite outgrowth. Thus, in AIM 3, we will provide a causal link between m6A-dependent dysregulation of TDP-43 and neurodegeneration by using CRISPR-Cas-based site-specific m6A editing of TDP-43 mRNA. Our work is expected to elucidate the role of m6A modification on TDP-43 subcellular distribution and functions. Thus, our findings will have a positive impact by advancing our mechanistic understanding of TDP-43 requirement for neuronal survival and by providing new targets for epitranscriptome- modulating therapies for age-related TDP-43 proteinopathies.

概括 痴呆性神经退行性疾病影响数百万人，每年造成的损失超过 3800 亿美元 美元仅在美国。深入了解这些不治之症的潜在有效治疗策略 疾病，在此我们将注意力集中在 RNA 结合蛋白 TAR DNA 结合蛋白-43 (TDP-43)， 其亚细胞分布和作用的改变被认为导致了许多痴呆症 包括阿尔茨海默病 (AD) 和肌萎缩侧索硬化症 - 额颞叶的临床谱 痴呆症（ALS/FTD）。 TDP-43 与其他具有低复杂性结构域的 RNA 结合蛋白一样，参与 通过液-液相分离形成与 RNA 加工相关的无膜细胞器 （LLPS）组装。虽然 TDP-43 的疾病相关突变可以破坏 LLPS 及其随后的作用，但如何 野生型 TDP-43 亚细胞分布和功能在大多数 AD 和 ALS/FTD 病例中发生改变，这 基因突变呈阴性，仍然是个谜。在此，我们建议研究转录后的作用 N6-甲基腺苷 (m6A) 介导的 TDP-43 调节机制。这个想法源于我们的发现 TARDBP（编码 TDP-43 的基因）是胚胎干细胞衍生运动中的 m6A 靶标之一 神经元（ES-MN），其表达受到 m6A 表观转录组调控。我们的中心假设是 TDP-43 上 m6A 标记的丢失会导致其表达及其亚细胞分布和作用发生变化， 这反过来又可能导致神经退行性变。这项研究的基本原理是，一旦知道如何 m6A 介导的 TDP-43 调节有助于神经变性、创新遗传或药理学 可以制定治疗这些人类无法治愈疾病的策略。我们建议首先（AIM 1） 表征 MN 对 m6A 丢失的时空敏感性。据此，我们将定义MN的模式 Mettl3 缺失的小鼠在症状前和症状期均出现脊髓和脑干损失 使用一系列行为、生理和形态学技术的阶段。那么，自从我们初步 数据显示，当 m6A 丢失时，TARDBP 水平会增加，在 AIM 2 中，我们将确定 m6A 的丢失如何 损害 TDP-43 表达及其亚细胞定位。我们的初步数据还表明，m6A 耗尽 ES-MN 会导致存活率和神经突生长减少。因此，在 AIM 3 中，我们将提供之间的因果关系 使用基于 CRISPR-Cas 的位点特异性 m6A 导致 TDP-43 的 m6A 依赖性失调和神经变性 TDP-43 mRNA 的编辑。我们的工作有望阐明 m6A 修饰对 TDP-43 亚细胞的作用 分布和功能。因此，我们的发现将通过推进我们的机制产生积极影响 了解 TDP-43 对神经元存活的要求并为表观转录组提供新的靶标 与年龄相关的 TDP-43 蛋白病的调节疗法。