METTL3-mediated regulation of motor neuron function

摘要 痴呆性神经退行性疾病影响数百万人，每年的成本超过3800亿美元 仅在美国就有美元。洞察这些不治之症的潜在有效治疗策略 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)上， 其亚细胞分布和作用的改变被认为是导致许多痴呆障碍的原因 包括阿尔茨海默病(AD)和肌萎缩侧索硬化症的临床谱-额颞部 痴呆(ALS/FTD)。与其他具有低复杂结构域的RNA结合蛋白一样，TDP-43参与了 液-液分离形成与RNA加工相关的无膜细胞器 (LLP)组件。虽然TDP-43中的疾病相关突变可以扰乱LLP及其随后的作用，但如何 AD和ALS/FTD患者主要表现为野生型TDP-43亚细胞分布和功能改变。 基因突变是否为阴性，仍然是个谜。在此，我们建议研究转录后蛋白的作用。 N6-甲基腺苷(M6A)介导的TDP-43的调控机制这个想法源于我们的发现 编码tdp-43的基因TARDBP是胚胎干细胞衍生马达的m6A靶点之一。 神经元(ES-MN)，其表达受m6A表位转录调控。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的变化， 反过来，这可能会导致神经退化。这项研究的基本原理是，一旦知道如何 M6A介导的TDP-43调节有助于神经退行性变、创新的遗传或药理学 可以制定出治疗这些无法治愈的人类疾病的策略。我们建议首先(目标1) 表征MNS对m6A丢失的时空敏感性。相应地，我们将定义MN的模式 症状前和症状时METTL3缺乏症小鼠脊髓和脑干的丢失 使用一系列的行为、生理和形态技术。然后，从我们的预赛开始 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丢失的方式 损害TDP-43的表达及其亚细胞定位。我们的初步数据还显示，M6A在 ES-MNS导致存活率降低和轴突生长。因此，在目标3中，我们将提供 基于CRISPR-Cas的位点特异性M6A依赖的TDP-43调节失调和神经变性 TDP-43mRNA的编辑。我们的工作有望阐明M6A修饰在TDP-43亚细胞中的作用 分布和功能。因此，我们的发现将通过推进我们的机械论产生积极的影响 了解TDP-43对神经元存活的要求，并为表位转录组学提供新的靶点。 与年龄相关的TDP-43蛋白病的调节治疗。

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.