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

TAR DNA结合蛋白-43 （TDP-43）是一种关键的RNA结合蛋白，密切参与RNA代谢的许多方面。虽然主要定位于细胞核，但TDP-43穿梭于细胞核和细胞质之间，发挥其生理功能。作为一种易于聚集的蛋白，已知TDP-43在细胞的细胞质中聚集并形成朊病毒样固体聚集体，导致核TDP-43的隔离。TDP-43的这种行为已被确定为神经退行性疾病谱系的病理标志，包括肌萎缩侧索硬化症和额颞叶痴呆（ALS/FTD），并已在阿尔茨海默病和相关痴呆中被描述。病理细胞质TDP-43内含物被假设通过核耗竭和细胞质聚集参与疾病的发病机制。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。我们实验室最近的研究表明，在C9orf72 ALS/FTD的多个脑区中存在异常的RNA A-I编辑，我们检测到A-I编辑的双向变化。从那时起，我们产生的初步数据表明，TDP-43的核输出可以通过腺苷脱氨酶作用于双链RNA （ADAR）介导的A-I RNA编辑来调节。我们发现，通过ADAR2在哺乳动物细胞系中过表达来增强RNA A-I编辑，可以诱导TDP-43易位到需要TDP-43功能RNA结合域的细胞质上。相反，过表达无催化活性的ADAR2不会改变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依赖机制诱导TDP-43细胞质错定位。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类诱导多能干细胞（iPSC），以进行a - i编辑介导的TDP-43核输出。我们将在表达多西环素诱导的et- on ADAR2构建物的iPSC-MNs中验证a - i RNA编辑介导的TDP-43细胞质积累：野生型ADAR2、催化无活性ADAR2 （ADAR2E396A）和催化超活性ADAR2 （ADAR2E488Q）。为了解决rna编辑诱导的TDP-43错定位对TDP-43功能的影响，我们将检查TDP-43内含物的疾病相关特征（ai1）。为了确定与TDP-43结合的mrna的身份，并且可能是A-I RNA编辑介导的错误定位所必需的，我们将对Aim1中描述的经过低编辑和超编辑的iPSC-MNs进行eCLIP-seq。此外，我们将在C9orf72 iPSC-MNs中执行eCLIP，以比较rna编辑诱导的TDP-43结合转录本与内源性疾病相关的转录本（目的2）。最后，在Aim 3中，我们将使用稳定转导的SH-SY5Y细胞，表达上述多西环素诱导的Tet-On ADAR2构建体，对TDP-43核输出新途径中涉及的分子和细胞机制进行探索性研究。

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.