RNA Surveillance and Protein Translation in FTD

额颞性痴呆(FTD)是一种与年龄相关的非阿尔茨海默病，以进行性痴呆为特征 额叶和颞叶的神经元丢失。FTD的一个子集由蛋白质包涵体的病理学定义 融合肉瘤(FUS)阳性，故命名为FUS-FTD。FTD与肌萎缩侧索硬化症(ALS) 具有广泛的临床、病理和遗传特征。FUS致病突变可导致ALS和 FTD和FUS蛋白病也可在散发性疾病中检测到。FUS主要在原子核内，但 具有致病突变的蛋白质错误定位并在细胞质中积累。蛋白质聚集体 由FUS突变引起的可能会隔离其他蛋白质以损害细胞功能，导致受损 神经元。我们最近发现，FUS的FTD/ALS突变抑制了蛋白质翻译，并高度激活了mRNAs的无义介导的衰退(NMD)。在这个项目中要检验的假设是 蛋白质翻译和信使核糖核酸监测的失调导致FUS-FTD中皮质神经元的丢失。 设计了三个特定的目标来使用体外和动物模型以及FTD来验证这一假说 病人的组织。目的1是确定致病因子是如何干扰mRNANMD和蛋白质翻译的。 FTD小鼠模型和患者组织中的突变。我们将确定NMD因子和翻译相关蛋白是否被隔离在R521G FUS转基因小鼠前脑神经元的FUS包涵体中 不同的年龄。我们将测量R521G FUS小鼠的mRNA周转率和蛋白质翻译效率 这些扰动与神经元功能障碍和FTD疾病进展相关。此外，我们还将审查 NMD因子和蛋白质翻译蛋白是否被隔离在FTD患者组织中。目标2是确定 致病FUS抑制FTD小鼠的特异性蛋白。我们将在FTD小鼠身上应用嘌呤霉素标记 并使用蛋白质组学的方法来确定突变型FUS对前脑蛋白质翻译的影响 神经元。此外，主动翻译的mRNAs将使用RNA-Seq在多聚体部分中进行识别和定量。来自组学方法的结果将被整合起来进行途径分析，以揭示致病 FUS损伤特定途径中的蛋白质，为FTD的病因学提供了新的见解。目标3是为了阐明 Rna结合和翻译后修饰在致病性FUS功能障碍中的意义。我们会 使用光遗传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意义。我们最近 发现FUS在对RNA结合至关重要的残基上被乙酰化，我们将测试乙酰化无效和模仿 突变会影响FUS包涵体、NMD和蛋白质翻译。此外，我们还将审查是否 致病的FUS形成依赖于RNA和不依赖于RNA的包涵体，对 神经元。拟议的实验将彻底检查一种新的疾病机制，使用创新的 接近了。我们提议的工作的完成将有助于阐明FUS FTD的分子机制。

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.