RNA Surveillance and Protein Translation in FTD

FTD 中的 RNA 监测和蛋白质翻译

• 批准号: 10449486
• 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
• 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/10449486
• 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

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（Frontotemporal dementia，FTD）是一种与年龄相关的非阿尔茨海默性痴呆， 额颞叶的神经元缺失FTD的一个子集定义为蛋白夹杂物的病理学 融合肉瘤（FUS）阳性，因此命名为FUS-FTD。FTD和肌萎缩侧索硬化症（ALS）的份额 广泛的临床、病理和遗传特征。FUS的致病性突变导致ALS和 FTD和FUS蛋白质病也在散发性疾病中检出。FUS主要在细胞核中，但 具有致病突变的蛋白质在细胞质中错误定位和积累。蛋白质聚集体 FUS突变诱导的蛋白质可能会螯合其他蛋白质，从而损害细胞功能， 神经元我们最近发现FUS的FTD/ALS突变抑制蛋白质翻译并过度激活mRNA的无义介导的衰变（NMD）。在这个项目中要检验的假设是， 蛋白质翻译和mRNA监视的失调导致FUS-FTD中皮质神经元丢失。 设计了三个特定目的，以使用体外和动物模型以及FTD检验假设 患者组织。目的1是确定致病性FUS如何干扰mRNA NMD和蛋白质翻译 FTD小鼠模型和患者组织中的突变。我们将确定在R521 G FUS转基因小鼠中，NMD因子和防御相关蛋白是否被隔离在前脑神经元中的FUS包涵体中。 不同年龄我们将测量R521 G FUS小鼠中的mRNA周转率和蛋白质翻译效率， 将扰动与神经元功能障碍和FTD疾病进展相关。此外，我们将研究 NMD因子和蛋白翻译蛋白是否被隔离在FTD患者组织中。目标2：识别 FTD小鼠中致病性FUS抑制的特异性蛋白质。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鉴定前脑中突变FUS影响的蛋白质翻译变化 神经元此外，将使用RNA-Seq在多核糖体组分中鉴定和定量主动翻译的mRNA。组学方法的结果将被整合用于途径分析，以揭示致病性是否 FUS损害特定途径中的蛋白质，为FTD病因学提供了新的见解。目的3是阐明 RNA结合和翻译后修饰在致病性FUS功能障碍中的意义。我们将 在光遗传学Cry 2 olig-FUS-mCherry系统中使用一组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意义。我们最近 我们发现FUS在RNA结合的关键残基处被乙酰化，我们将测试乙酰化-无效和-模拟 突变影响FUS内含物、NMD和蛋白质翻译。此外，我们亦会研究 致病性FUS形成RNA依赖性和非依赖性包涵体，其产生不同水平的毒性， 神经元拟议的实验将彻底研究一种新的疾病机制， 接近。完成我们拟定的工作将有助于阐明FUS FTD的分子机制。