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

• 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
• 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' s Disease; Alzheimer' 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

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）是一种重要的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在细胞的细胞质中积累并形成朊病毒样固体聚集体，导致核TDP-43的隔离。TDP-43的这种行为已被充分确立为包括肌萎缩侧索硬化和额颞叶痴呆（ALS/FTD）的神经变性疾病谱的病理学标志，并且已在阿尔茨海默病和相关痴呆中描述。病理性胞质TDP-43包涵体已被假设为通过核耗竭和胞质聚集两者促成疾病发病机制。尽管进行了广泛的研究，但在疾病条件下引发这种病理学的机制仍然难以捉摸。我们实验室最近的研究表明，异常的RNA A-I编辑存在于C9 orf 72 ALS/FTD的多个脑区，我们检测到A-I编辑的双向变化。从那时起，我们已经产生了初步的数据，表明TDP-43核输出可以通过腺苷脱氨酶作用于双链RNA（阿达尔）介导的A-I RNA编辑来调节。我们表明，在哺乳动物细胞系中通过ADAR 2过表达增强RNA A-I编辑诱导TDP-43易位到细胞质，需要TDP-43的功能性RNA结合结构域。相反，催化失活的ADAR 2的过表达不改变TDP-43的核定位。这些发现使我们假设A-I编辑的异常增加通过RNA依赖性机制诱导TDP-43细胞质错误定位。为了确定这种编辑诱导的TDP-43核输出是否也发生在神经元环境中，我们将扩展我们的初步数据，并检查分化为运动神经元的人诱导多能干细胞（iPSC）的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型Tet-On ADAR 2构建体的iPSC-MN中的细胞质积累：野生型ADAR 2、催化失活的ADAR 2（ADAR 2 E396 A）和催化超活性的ADAR 2（ADAR 2 E488 Q）。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包含物的疾病相关特征（Aim 1）。为了确定与TDP-43结合并且可能是A-I RNA编辑介导的错误定位所必需的mRNA的身份，我们将对iPSC-MN进行eCLIP-seq，iPSC-MN如Aiml中所述被遗传改变以进行hypo和hyper-editing。此外，我们将在C9 orf 72 iPSC-MN中进行eCLIP，以比较RNA编辑诱导的TDP-43结合转录物与内源性疾病相关的转录物（目的2）。最后，在目的3中，我们将使用表达上述多西环素诱导的Tet-On ADAR 2构建体的稳定转导的SH-SY 5 Y细胞，对TDP-43核输出的这种新途径中涉及的分子和细胞机制进行探索性研究。