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 痴呆的神经退行性疾病会影响数百万的人，并且每年成本超过3800亿美元 仅在美国的美元。洞悉这些无法治愈的潜在有效治疗策略 疾病，我们将注意力集中在RNA结合蛋白TAR DNA结合蛋白-43（TDP-43）上， 细胞的分布和作用的改变被认为会导致多种痴呆症 包括阿尔茨海默氏病（AD）和肌萎缩性侧索硬化症的临床光谱 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 与RNA处理相关的无膜细胞器的形成通过液体液相分离 （llps）组件。尽管TDP-43中与疾病相关的突变会破坏LLP及其随之而来的角色，但如何如何 在AD和ALS/FTD病例中，野生型TDP-43亚细胞分布和功能发生了变化，它们 是基因突变阴性，保持神秘。我们在本文中建议调查转录后的作用 由N6-甲基腺苷（M6A）介导的TDP-43的调节机制。这个想法源于我们的发现 TARDBP是TDP-43的基因编码，是胚胎干细胞衍生电动机的M6A靶标之一 神经元（ES-MNS），其表达在M6a的表面转录组调节下。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的变化， 反过来，这可能有助于神经变性。这项研究的理由是，一旦知道了 M6A介导的TDP-43调节有助于神经变性，创新遗传或药物 可以制定策略来治疗这些无法治愈的人类疾病。我们建议首先（目标1） 表征MNS对M6A损失的时空敏感性。彼此之间，我们将定义MN的模式 METTL3部署的小鼠脊髓和脑干的损失均在Presymptom和症状下损失 使用一系列行为，物理和形态学技术的阶段。然后，由于我们的初步 数据表明，当M6A丢失时TARDBP水平会增加，在AIM 2中，我们将确定M6A的损失如何 损害TDP-43表达及其亚细胞定位。我们的初步数据还表明M6A部署 ES-MNS导致生存率降低和神经落生。在AIM 3中，我们将提供一个因果关系 通过使用基于CRISPR-CAS的特定地点M6A，TDP-43和神经变性的M6A依赖性失调 TDP-43 mRNA的编辑。我们的工作有望阐明M6A修饰在TDP-43亚细胞上的作用 分布和功能。那就是我们的发现将通过提高我们的机制来产生积极的影响 了解TDP-43对神经元生存的要求，并通过为表面翻译组提供新的目标 调节与年龄相关的TDP-43蛋白质病的疗法。