The role of ADAR2-associated RNA editing in pathogenesis of ALS

ADAR2 相关 RNA 编辑在 ALS 发病机制中的作用

• 批准号: 10084222
• 负责人: STELLA DRACHEVA
• 金额: --
• 依托单位: JAMES J PETERS VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-10-01 至 2021-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10084222
• 关键词: ALS patients; AMPA Receptors; Acids; Age; Amyotrophic Lateral Sclerosis; Animals; Astrocytes; Autopsy; Biological Assay; Brain region; C9ORF72; CRISPR/Cas technology; Calcium; Cells; Coculture Techniques; DRADA2b protein; Data; Disease; Disease Pathway; Disease Progression; Down-Regulation; Enzymes; Event; Family history of; Fluorescence-Activated Cell Sorting; Gene Expression; Genes; Glutamates; Goals; Healthcare Systems; Heterogeneity; Human; Immunohistochemistry; Inflammatory; Inflammatory Response; Inherited; Kainic Acid Receptors; Knowledge; Laboratories; Lasers; Massive Parallel Sequencing; Measures; Mediating; Messenger RNA; Methods; Microglia; Molecular; Motor Cortex; Motor Neurons; Muscle Weakness; Mutation; Nature; Neurodegenerative Disorders; Neurons; Nuclear Antigens; Occipital lobe; Oligodendroglia; Paralysed; Pathogenesis; Pathologic; Patients; Population; Potassium; Process; Publishing; Quality of life; RNA Editing; Reporting; Research; Risk Factors; Role; Serotonin Receptor 5-HT2C; Signal Transduction; Specimen; Spinal; Spinal Cord; Spinal cord injury; System; Testing; Time; Tissues; Transcript; Transcription Alteration; Variant; Veterans; base; cell type; cytokine; differential expression; improved; induced pluripotent stem cell; innovation; male; military service; neuroinflammation; neuronal excitability; novel; nucleocytoplasmic transport; receptor; restriction enzyme; sex; sporadic amyotrophic lateral sclerosis; stem; superoxide dismutase 1; transcriptome; transcriptome sequencing; transmission process

Amyotrophic Lateral Sclerosis (ALS) is a devastating and fatal neurodegenerative disease that is characterized by progressive muscle weakness and eventual paralysis. The known risk factors for ALS are increasing age and male sex; recent data also implicate military service, thus putting an additional burden on the VA healthcare system. Approximately 90% of all ALS cases are classified as sporadic ALS (sALS) with no family history of the disease. The remaining cases are inherited in a dominant fashion, and mutations in ~30 genes (most commonly in C9orf72, SOD1, TARDBP, FUS) are currently known to cause ALS. Hyperexcitability that stems from pathophysiological disturbance of glutamatergic transmission has been reported in all types of ALS cases, and may be a common disease pathway that predisposes motoneurons to degeneration. It has been suggested that the glutamatergic (Glu) deficit in ALS may result from altered function of the AMPA Glu receptors due to inefficient RNA editing of the GluA2 AMPA subunit. The GluA2 editing is catalyzed by the editing enzyme ADAR2. Our data, as well as data published by others, showed the downregulation of ADAR2 and inefficient editing of the GluA2 in the motor cortex of patients with C9orf72-associated ALS (C9 ALS) as well as in spinal motoneurons of patients with sALS. We also reported the downregulation of ADAR2 and the consequent decrease of editing in several of its targets in animals subjected to spinal cord injury (SCI). Moreover, in SCI these events are triggered by inflammatory response and result in changes in gene expression that are likely to contribute to post-SCI motoneuron hyperexcitability. Based on these data and the fact that, as in SCI, neuroinflammation is present in ALS, we hypothesize that: (1) ADAR2 is downregulated in both C9 ALS and sALS, leading to alterations in RNA editing in its targets. In particular, in addition to GluA2, ADAR2 editing of other molecules which are important for neuronal excitability [such as, GluA3-4, kainate receptor subunits GluK1-2, serotonin 2C receptor (5-HT2CR), as well as potassium (Kv1.1) and calcium (Cav1.3) channels] is also altered in C9 ALS and sALS; (2) The downregulation of ADAR2 in ALS results from several different pathological features associated with the disease. Whereas the decrease of ADAR2 in sALS is triggered by neuroinflammation, the availability of the functional ADAR2 in C9 ALS is also influenced by its cytoplasmic retention, which is caused by a disruption of nucleocytoplasmic transport. We will test these hypotheses by characterizing ALS-associated alterations (1) in the ADAR2 expression and localization and (2) in editing of the transcripts that are targeted by ADAR2. These studies will be performed in laser microdissected neurons obtained from autopsy tissues of patients with C9 and sporadic ALS (Aim1) as well as in motoneurons differentiated from human induced pluripotent stem cells (hiPSC) derived from ALS patients (Aim2). In addition, previously published studies of ALS-associated transcriptional alterations employed whole (cellular heterogeneous) tissue specimens. However, because of the heterogeneity of cell types in the CNS, accurate assessment of the ALS transcriptome cannot be inferred from the data obtained from these specimens that combine signals from all cell types. Therefore, we will study cell- type-specific transcriptomes in C9 ALS and sALS (Aim3). These studies are enabled by the novel methods recently developed in our lab, and should enhance the likelihood of elucidating disease relevant variations, including those related to ADAR2 and editing. Overall, this is an innovative proposal that builds on the interdisciplinary nature of our collaborative team to investigate RNA editing changes that characterize ALS and to define unique changes occurring only in C9 or sporadic ALS. Our goal is to advance the current knowledge and to discover new treatments.

肌萎缩侧索硬化症（ALS）是一种破坏性和致命的神经退行性疾病， 以逐渐的肌肉无力和最终的瘫痪为特征。ALS的已知危险因素包括 年龄和男性性别的增加;最近的数据也涉及服兵役，从而增加了妇女的负担。 VA医疗保健系统大约90%的ALS病例被归类为散发性ALS（sALS）， 疾病的家族史其余病例以显性方式遗传，约30%的病例发生突变。 基因（最常见于C9 orf 72、SOD 1、TARDBP、FUS）目前已知引起ALS。 兴奋过度，源于病理生理障碍的血管紧张素转换酶， 在所有类型的ALS病例中都有报道，可能是一种常见的疾病途径， 运动神经元退化。有人认为ALS中的谷氨酸（Glu）缺陷可能导致 由于GluA 2 AMPA亚基的低效RNA编辑，AMPA Glu受体的功能改变。 GluA 2编辑由编辑酶ADAR 2催化。我们的数据，以及其他人发布的数据， 显示ADAR 2的下调和GluA 2在运动皮层的无效编辑， C9 orf 72相关ALS（C9 ALS）以及sALS患者的脊髓运动神经元中。我们还报道 ADAR 2的下调以及随之而来的在动物中几个靶点的编辑减少 脊髓损伤（SCI）。此外，在SCI中，这些事件是由炎症反应触发的， 并导致基因表达的变化，这些变化可能导致SCI后运动神经元过度兴奋。 基于这些数据和事实，如SCI，神经炎症存在于ALS，我们假设 （1）ADAR 2在C9 ALS和sALS中均下调，导致其靶点的RNA编辑发生改变。 特别地，除了GluA 2之外，ADAR 2编辑对神经元细胞重要的其他分子， 兴奋性[如GluA 3 -4、红藻氨酸受体亚单位GluK 1 -2、5-羟色胺2C受体（5-HT 2CR），以及 钾（Kv1.1）和钙（Cav1.3）通道]也在C9 ALS和sALS中改变;（2）下调 ALS中ADAR 2的表达由与疾病相关的几种不同病理特征引起。而 sALS中ADAR 2的减少是由神经炎症触发的，C9中功能性ADAR 2的可用性 ALS还受到其细胞质滞留的影响，这是由核细胞质的破坏引起的。 运输 我们将通过描述ADAR 2中ALS相关改变（1）来测试这些假设 表达和定位以及（2）编辑ADAR 2靶向的转录物。这些研究将 在从C9和散发性C9患者的尸检组织中获得的激光显微切割神经元中进行 ALS（Aim 1）以及从人诱导多能干细胞（hiPSC）分化的运动神经元 来自ALS患者（Aim 2）。此外，以前发表的ALS相关转录的研究， 改变采用全（细胞异质）组织样本。但因为 由于CNS中细胞类型的异质性，ALS转录组的准确评估不能从 从这些标本中获得的数据结合了来自所有细胞类型的联合收割机信号。因此，我们将研究细胞- C9 ALS和sALS中的类型特异性转录组（Aim 3）。这些研究是由新的方法， 最近在我们的实验室开发，并应提高阐明疾病相关变异的可能性， 包括与ADAR 2和编辑相关的那些。 总的来说，这是一个创新的建议，建立在我们的合作的跨学科性质， 研究团队调查表征ALS的RNA编辑变化，并定义仅在ALS中发生的独特变化。 C9或散发性ALS。我们的目标是推进现有知识并发现新的治疗方法。

项目成果

Divergent single cell transcriptome and epigenome alterations in ALS and FTD patients with C9orf72 mutation.

• DOI: 10.1038/s41467-023-41033-y
• 发表时间: 2023-09-15
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Li, Junhao;Jaiswal, Manoj K.;Chien, Jo-Fan;Kozlenkov, Alexey;Jung, Jinyoung;Zhou, Ping;Gardashli, Mahammad;Pregent, Luc J.;Engelberg-Cook, Erica;Dickson, Dennis W.;Belzil, Veronique V.;Mukamel, Eran A.;Dracheva, Stella
• 通讯作者: Dracheva, Stella