RNA Surveillance and Protein Translation in FTD

FTD 中的 RNA 监测和蛋白质翻译

基本信息

批准号：
10455737
负责人：
Haining Zhu
金额：
$ 53.85万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10455737
关键词：
Acetylation Affect Age Amyotrophic Lateral Sclerosis Animal Model Cell Culture Techniques Cell Nucleus Cell model Cell physiology Clinical Cultured Cells Cytoplasm Disease Disease Progression Etiology Frontotemporal Dementia Functional disorder Future Genetic Impairment In Vitro Label Light Liquid substance Lobe Measures Mediating Membrane Messenger RNA Methods Molecular Mus Mutation Names Nerve Degeneration Neuronal Dysfunction Neurons Nonsense-Mediated Decay Pathogenesis Pathogenicity Pathologic Pathology Pathway Analysis Pathway interactions Patients Phase Play Polyribosomes Post-Translational Protein Processing Process Prosencephalon Proteins Proteomics Protocols documentation Publishing Puromycin RNA RNA Binding Research Ribonucleoproteins Role System Temporal Lobe Testing Tissues Toxic effect Transgenic Mice Translating Translations Work age related base cohort design disease-causing mutation experimental study frontal lobe frontotemporal lobar dementia-amyotrophic lateral sclerosis fused in sarcoma innovation insight interdisciplinary approach mRNA Decay mRNA Surveillance mouse model mutant neuron loss neurotoxicity nonalzheimer dementia novel optogenetics polysome profiling protein aggregation spatiotemporal therapeutic development transcriptome sequencing

项目摘要

Frontotemporal dementia (FTD) is an age-related non-Alzheimer dementia characterized by progressive neuronal loss in the frontotemporal lobes. A subset of FTD is defined by the pathology of protein inclusions positive for Fused in Sarcoma (FUS), thus named FUS-FTD. FTD and amyotrophic lateral sclerosis (ALS) share a wide spectrum of clinical, pathological and genetic features. Pathogenic mutations of FUS cause both ALS and FTD, and FUS proteinopathy is also detected in sporadic diseases. FUS is primarily in the nucleus, but the protein with a disease-causing mutation mislocalizes and accumulates in the cytoplasm. Protein aggregates induced by FUS mutations may sequester other proteins to compromise cellular functions, resulting in impaired neurons. We recently showed that FTD/ALS mutations of FUS suppressed protein translation and hyper-activated the nonsense-mediated decay (NMD) of mRNAs. The hypothesis to be tested in this project is that the dysregulation of protein translation and mRNA surveillance contributes to cortical neuron loss in FUS-FTD. Three specific aims are designed to test the hypothesis using in vitro and animal models as well as FTD patient tissues. Aim 1 is to determine how mRNA NMD and protein translation are perturbed by pathogeneic FUS mutations in FTD mouse models and patient tissues. We will determine whether NMD factors and translation-related proteins are sequestered in FUS inclusions in forebrain neurons in R521G FUS transgenic mice at different ages. We will measure mRNA turnover rates and protein translation efficiency in R521G FUS mice and correlate the perturbations to neuronal dysfunction and FTD disease progression. Moreover, we will examine whether NMD factors and protein translation proteins are sequestered in FTD patient tissues. Aim 2 is to identify specific proteins suppressed by pathogenic FUS in FTD mice. We will apply the puromycin labeling in FTD mice and use the proteomic approach to identify changes in protein translation impacted by mutant FUS in forebrain neurons. In addition, actively translated mRNAs will be identified and quantified in polysome fractions using RNA-Seq. Results from the –omics approaches will be integrated for pathway analysis to reveal whether pathogenic FUS impairs proteins in specific pathways, providing novel insights into the FTD etiology. Aim 3 is to elucidate the significance of RNA binding and post-translational modifications in the dysfunction of pathogenic FUS. We will use a cohort of RNA binding-deficient mutations in an optogenetic Cry2olig-FUS-mCherry system to examine the significance of RNA binding in FUS inclusion formation and dysfunction in cortical neurons. We recently found FUS is acetylated at residues critical to RNA binding, we will test how acetylation-null and -mimicking mutations affect FUS inclusions, NMD and protein translation. In addition, we will also examine whether pathogenic FUS forms RNA-dependent and -independent inclusions that produce different levels of toxicity to neurons. The proposed experiments will thoroughly examine a novel disease mechanism using innovative approaches. Completion of our proposed work will help elucidate molecular mechanisms underlying FUS FTD.

额颞叶痴呆 (FTD) 是一种与年龄相关的非阿尔茨海默氏痴呆，其特征是进行性进展 FTD 的一个子集是由蛋白质内含物的病理学定义的。肉瘤融合 (FUS) 呈阳性，因此命名为 FUS-FTD，与肌萎缩侧索硬化症 (ALS) 相同。 FUS 的致病突变具有广泛的临床、病理和遗传特征，可导致 ALS 和 ALS。 FTD 和 FUS 蛋白病也可在散发性疾病中检测到，FUS 主要发生在细胞核中，但 FUS 蛋白病主要发生在细胞核中。具有致病突变的蛋白质错误定位并积聚在细胞质中。 FUS 突变引起的突变可能会隔离其他蛋白质以损害细胞功能，从而导致细胞功能受损我们最近表明 FUS 的 FTD/ALS 突变会抑制蛋白质翻译并过度激活 mRNA 的无义介导的衰变 (NMD)。蛋白质翻译和 mRNA 监测的失调导致 FUS-FTD 中皮质神经元的丢失。设计了三个具体目标来使用体外和动物模型以及 FTD 来检验该假设目标 1 是确定 mRNA NMD 和蛋白质翻译如何受到致病性 FUS 的干扰。我们将在 R521G FUS 转基因小鼠的前脑神经元中确定 NMD 因子和翻译相关蛋白是否被隔离在 FUS 内含物中。我们将测量 R521G FUS 小鼠和不同年龄的 mRNA 周转率和蛋白质翻译效率。此外，我们将检查扰动与神经元功能障碍和 FTD 疾病进展的关系。目标 2 是确定 NMD 因子和蛋白质翻译蛋白是否被隔离在 FTD 患者组织中。 FTD 小鼠中致病性 FUS 抑制的特定蛋白质我们将在 FTD 小鼠中应用嘌呤霉素标记。并使用蛋白质组学方法来识别受前脑突变 FUS 影响的蛋白质翻译变化此外，将使用 RNA-Seq 方法对多核糖体组分中活跃翻译的 mRNA 进行鉴定和定量，并将其整合用于通路分析，以揭示是否具有致病性。 FUS 损害特定途径中的蛋白质，为 FTD 病因学提供新的见解。目标 3 是阐明 FTD 的病因。 RNA 结合和翻译后修饰在致病性 FUS 功能障碍中的重要性。使用光遗传学 Cry2olig-FUS-mCherry 系统中的一组 RNA 结合缺陷突变来检查 RNA 结合在皮质神经元 FUS 包涵体形成和功能障碍中的重要性。发现 FUS 在对 RNA 结合至关重要的残基上被乙酰化，我们将测试如何乙酰化无效和模拟此外，我们还将检查突变是否会影响 FUS 内含物、NMD 和蛋白质翻译。致病性 FUS 形成依赖于 RNA 和不依赖于 RNA 的包涵体，对人体产生不同程度的毒性。拟议的实验将利用创新技术彻底检查一种新的疾病机制。完成我们提出的工作将有助于阐明 FUS FTD 的分子机制。