Stress Granules and the Biology of TDP-43

应激颗粒和 TDP-43 的生物学

基本信息

批准号：
8432065
负责人：
Benjamin L Wolozin
金额：
$ 58.2万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-03-01 至 2016-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8432065
关键词：
Address Amyotrophic Lateral Sclerosis Binding Biological Biology Brain Cell Line Cell Nucleus Cell model Cells Chimera organism Complex Cytoplasm Cytoplasmic Granules Cytoplasmic Inclusion Data Dendrites Disease Dyes Elements Employee Strikes Familial Amyotrophic Lateral Sclerosis Frontotemporal Dementia Functional disorder Genes Genotoxic Stress Growth Factor Hippocampus (Brain)Human Human Cell Line Image Knock-out Label Lead Length Link Location Measures Mediating Messenger RNA Molecular Monitor Motor Motor Neurons Mus Mutation Nerve Degeneration Neurons Nuclear RNA Pathology Pathway interactions Polyribosomes Population Process Proliferating Protein Binding Proteins RNA RNA-Binding Proteins Regulation Research Role SCA2 protein Spinal Cord Stress Structure Synapses Therapeutic Intervention Tissues Toxic effect Transcript Transgenic Mice Translations Work cytotoxicity genetic risk factor human tissue in vivo induced pluripotent stem cell mutant neuronal cell body polyglutamine protein TDP-43 protein complex protein metabolism response trafficking

项目摘要

TDP-43 is the principle component of inclusions in amyotrophic lateral sclerosis (ALS) and in some frontotemporal dementia (FTLD-U). TDP-43 is a nuclear RNA binding protein, which translocates to the cytoplasm during stress where it forms cytoplasmic granules. Our research indicates that these cytoplasmic TDP-43 inclusions co-localize with RNA granules termed "stress granules" (SGs) in cell models and in the human brain. Disease-linked mutations in TDP-43 also increase formation of inclusions associated with SGs. These data point to a strong biological connection between SGs and TDP-43. This proposal will address the role of SG-dependent and independent processes in the pathophysiology of ALS. We hypothesize that SG biology stimulates formation of TDP-43 inclusions, and that pathogenic factors linked to ALS increase TDP-43 inclusion formation through a process mediated by SG pathways. Aim 1 will use induced pluripotent stem cells (IPSCs, generated from control and TDP-43 mutant human cell lines) and hippocampal neurons to characterize the regulation of TDP-43 inclusion formation. We will use imaging to determine how disease-linked mutations in TDP-43 modify formation and dispersion of RNA granules under basal or stressed conditions, including genotoxic stress (e.g., effects of ataxin-2 ¿ expanded polyglutamine regions), excitatory stress (K+) or growth factor stimulation. In each case we evaluate the role of RPCs (including SGs) in particular locations, such as the soma or dendritic arbor, by genetically restricting RPC formation to the nucleus, soma or soma/dendrite, and examining toxicity. Neurodegeneration will be monitored by measuring dendritic length under the different conditions, and putative changes in dendritic structure will be validated in human tissues. In Aim 2 we will identify molecular factors associated with TDP-43 inclusions. We will determine how pathological mutations in TDP-43 or other SG associated proteins modify the proteins and mRNA that associate with TDP-43 under conditions ¿ inclusions. In Aim 3 we will determine whether TDP-43 forms inclusion through a SG-augmented mechanism in vivo. This aim will apply the work of Aims 1 & 2 to the in vivo setting, using transgenic mice expressing WT TDP-43. We will identify proteins associated with inclusions in inducible TDP-43 WT transgenic mice. We will investigate examine transgenic mouse lines expressing mutant TDP-43 to determine whether expression of ataxin-2 Q21, 31 or 58 increases TDP-43 motor dysfunction, pathology. Finally we will determine whether ataxin-2 knockout inhibits TDP-43 pathology. Investigating the particular elements of the SG pathway that regulate TDP-43 inclusion formation will identify selective approaches for therapeutic intervention to delay or halt the progression of ALS. !

TDP-43 是肌萎缩侧索硬化症 (ALS) 和某些疾病中包涵体的主要成分额颞叶痴呆 (FTLD-43) 是一种核 RNA 结合蛋白，易位至我们的研究表明，在应激过程中，细胞质会形成细胞质颗粒。细胞质 TDP-43 内含物与细胞中称为“应激颗粒”(SG) 的 RNA 颗粒共定位模型和人脑中与疾病相关的 TDP-43 突变也会增加这些数据表明 SG 和 SG 之间存在很强的生物学联系。 TDP-43 该提案将讨论 SG 依赖和独立流程在 ALS 的病理生理学我们勇敢地承认 SG 生物学会刺激 TDP-43 包涵体的形成，与 ALS 相关的致病因素通过一个过程增加 TDP-43 包涵体的形成目标 1 将使用诱导多能干细胞（IPSC，由 SG 途径介导）。对照和 TDP-43 突变人类细胞系）和海马神经元来表征调节我们将使用成像技术来确定与疾病相关的突变是如何形成的。 TDP-43 在基础或应激条件下改变 RNA 颗粒的形成和分散，包括基因毒性应激（例如，ataxin-2 的影响“扩展的聚谷氨酰胺区域）、兴奋性应激在每种情况下，我们都会评估 RPC（包括 SG）的作用。特定位置，例如体细胞或树突乔木，通过基因限制 RPC 的形成细胞核、胞体或胞体/树突，并通过毒性检查来监测神经变性。测量不同条件下的树突长度，以及树突结构的推定变化将在人体组织中进行验证在目标 2 中，我们将鉴定与 TDP-43 相关的分子因子。我们将确定 TDP-43 或其他 SG 相关蛋白的病理突变是如何发生的。在一定条件下修改与 TDP-43 相关的蛋白质和 mRNA在目标 3 中，我们将确定 TDP-43 是否通过体内 SG 增强机制形成包涵体。将使用表达 WT TDP-43 的转基因小鼠将目标 1 和 2 的工作应用于体内环境。我们将鉴定与可诱导 TDP-43 WT 转基因小鼠中的内含物相关的蛋白质。研究检查表达突变 TDP-43 的转基因小鼠系以确定是否 ataxin-2 Q21、31 或 58 的表达会增加 TDP-43 运动功能障碍，最后我们将进行病理学分析。确定 ataxin-2 敲除是否抑制 TDP-43 病理学。调节 TDP-43 包涵体形成的 SG 途径的研究将确定选择性方法治疗干预以延缓或阻止 ALS 的进展。！