Mechanisms of A-I RNA editing-mediated nuclear export of TDP-43

A-I RNA编辑介导的TDP-43核输出机制

基本信息

批准号：
10575984
负责人：
Rita Sattler
金额：
$ 34.56万
依托单位：
ST. JOSEPH'S HOSPITAL AND MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-22 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10575984
关键词：
Active Biological Transport Address Adenosine Alzheimer&apos s Disease Alzheimer&apos s disease related dementia Behavior Biological Assay Brain region C9ORF72 Cell Line Cell Nucleus Cells Cellular Stress Characteristics Complex Cytomegalovirus Infections Cytoplasm Cytoplasmic Inclusion DRADA2b protein Data Data Set Dementia Diffusion Disease Double-Stranded RNA Doxycycline Drug Targeting Environment Event Functional disorder Funding Future Genetic Transcription Human Hyperactivity Immunoprecipitation Inosine Knowledge Laboratories Mammalian Cell Measures Mediating Messenger RNA Modeling Molecular Motor Neurons Mutation Neurodegenerative Disorders Neurons Nuclear Nuclear Envelope Nuclear Export Nuclear Pore Nuclear Protein Pathogenesis Pathologic Pathology Pathway interactions Phosphorylation Physiological Pore Proteins Proteins Publishing RNA RNA Binding RNA Editing RNA Processing RNA Recognition Motif RNA Splicing RNA Transport RNA metabolism RNA-Binding Proteins Research Role Solid Solubility Testing Tetanus Helper Peptide Time Transcript Translations Untranslated RNA Validation adenosine deaminase crosslink disease phenotype drug discovery exportin 1 protein exportin 5 frontotemporal lobar dementia-amyotrophic lateral sclerosis gain of function heterokaryon induced pluripotent stem cell innovation loss of function novel novel therapeutics overexpression patient population prion-like protein TDP-43 protein transport receptor stem cell differentiation stress granule trafficking transcriptome sequencing

项目摘要

TAR DNA binding protein – 43 (TDP-43) is a critical RNA binding protein that is intimately involved in many aspects of RNA metabolism. While primarily localized to the nucleus, TDP-43 shuttles between the nucleus and the cytoplasm performing its physiological functions. As an aggregation prone protein, TDP-43 is known to accumulate and from prion-like solid aggregates in the cytoplasm of cells leading to the sequestration of nuclear TDP-43. This behavior of TDP-43 has been well established as a pathological hallmark of a neurodegenerative disease spectrum encompassing amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) and has been described in Alzheimer’s disease and related dementias. Pathological cytoplasmic TDP-43 inclusions have been hypothesized to contribute to disease pathogenesis through both a nuclear depletion and the cytoplasmic aggregation. Despite extensive research, mechanisms that initiate this pathology under disease conditions remain elusive. Recent studies in our laboratory showed that aberrant RNA A-I editing is present in multiple brain regions of C9orf72 ALS/FTD, where we detected bidirectional changes in A-I editing. Since then, we have generated preliminary data suggesting that TDP-43 nuclear export can be regulated via Adenosine Deaminase Acting on double stranded RNA (ADAR)-mediated A-I RNA editing. We show that enhancing RNA A-I editing through ADAR2 overexpression in mammalian cell lines induces TDP-43 translocation to the cytoplasm requiring functional RNA binding domains of TDP-43. In contrast, the overexpression of catalytically inactive ADAR2 does not alter the nuclear localization of TDP-43. These findings led us to hypothesize that aberrant increases in A-I editing induces TDP-43 cytoplasmic mislocalization through an RNA dependent mechanism. To determine if this editing induced TDP-43 nuclear export also occurs in a neuronal environment, we will expand on our preliminary data and examine human induced pluripotent stem cell (iPSC) differentiated into motor neurons for A-I editing-mediated TDP-43 nuclear export. We will validate A-I RNA editing mediated cytoplasmic accumulation of TDP-43 in iPSC-MNs expressing doxycycline inducible Tet-On ADAR2 constructs: wildtype ADAR2, a catalytically inactive ADAR2 (ADAR2E396A) and a catalytically hyperactive ADAR2 (ADAR2E488Q). To address the effects of RNA-editing induced TDP-43 mislocalization on TDP-43 function, we will examine TDP-43 inclusions for disease-relevant characteristics (Aim1). To determine the identity of mRNAs bound to TDP-43 and potentially being necessary for A-I RNA editing-mediated mislocalization, we will perform eCLIP-seq on iPSC-MNs genetically altered for hypo and hyper-editing as described in Aim1. In addition, we will perform eCLIP in C9orf72 iPSC-MNs to compare RNA-editing induced TDP-43 bound transcripts to those associated with endogenous disease (Aim 2). Finally, in Aim 3, we will perform exploratory studies towards the identification of molecular and cellular mechanisms involved in this new pathway of TDP-43 nuclear export using stably transduced SH-SY5Y cells expressing the doxycycline-induced Tet-On ADAR2 constructs described above.

TAR DNA 结合蛋白 – 43 (TDP-43) 是一种关键的 RNA 结合蛋白，与 RNA 代谢的许多方面密切相关。虽然 TDP-43 主要定位于细胞核，但它在细胞核和细胞质之间穿梭，发挥其生理功能。作为一种易于聚集的蛋白质，已知 TDP-43 会在细胞的细胞质中积聚并形成朊病毒样固体聚集物，导致 TDP-43 被隔离在核中。TDP-43 的这种行为具有以下特点。已被确定为包括肌萎缩侧索硬化症和额颞叶痴呆 (ALS/FTD) 在内的神经退行性疾病谱系的病理标志，并在阿尔茨海默病和相关痴呆中得到描述，病理性细胞质 TDP-43 内含物已丰富，有助于疾病发病机制。尽管进行了广泛的研究，但在疾病条件下启动这种病理的机制仍然难以捉摸。异常的 RNA A-I 编辑存在于 C9orf72 ALS/FTD 的多个大脑区域，我们在其中检测到 A-I 编辑的双向变化，从那时起，我们生成了初步数据，表明 TDP-43 核输出可以通过作用于双链的腺苷脱氨酶来调节。我们发现，在哺乳动物细胞系中，通过 ADAR2 过表达来增强 RNA A-I 编辑会诱导 TDP-43 易位。相反，催化失活的 ADAR2 的过度表达不会改变 TDP-43 的核定位，这些发现使我们发现 A-I 编辑的异常增加会诱导 TDP-43 细胞质。为了确定这种编辑诱导的 TDP-43 核输出是否也发生在神经环境中，我们将扩展我们的初步数据并检查人类诱导的错误定位。多能干细胞 (iPSC) 分化为运动神经元，用于 A-I 编辑介导的 TDP-43 核输出我们将验证 A-I RNA 编辑介导的 TDP-43 在表达多西环素诱导的 Tet-On ADAR2 构建体的 iPSC-MN 中的细胞质积累：野生型 ADAR2，催化失活的 ADAR2 (ADAR2E396A) 和催化高活性的 ADAR2 (ADAR2E488Q)。为了解决 RNA 编辑诱导的 TDP-43 错误定位对 TDP-43 功能的影响，我们将检查 TDP-43 内含物的疾病相关特征 (Aim1)，以确定与 TDP-43 结合的 mRNA 的身份。并且对于 A-I RNA 编辑介导的错误定位可能是必要的，我们将在经过基因改造以进行低编辑和超编辑的 iPSC-MN 上进行 eCLIP-seq，如下所示此外，我们将在 C9orf72 iPSC-MN 中进行 eCLIP，以将 RNA 编辑诱导的 TDP-43 结合转录本与与内源性疾病相关的转录本进行比较（目标 2）。最后，在目标 3 中，我们将针对这些进行探索性研究。使用表达多西环素诱导的稳定转导的 SH-SY5Y 细胞，鉴定参与 TDP-43 核输出新途径的分子和细胞机制Tet-On ADAR2 构建体如上所述。