Stress Granules and the Biology of TDP-43

应激颗粒和 TDP-43 的生物学

• 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
• 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

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是肌萎缩性侧索硬化症（ALS）的夹杂物的原理成分，在某些中 额颞痴呆（FTLD-U）。 TDP-43是一种核RNA结合蛋白，可易位 在应激中形成细胞质颗粒的压力中的细胞质。我们的研究表明这些 细胞质TDP-43夹杂物与称为“应力颗粒”（SGS）的RNA颗粒共定位在细胞中 模型和人脑中。 TDP-43中疾病连接的突变也增加了 与SG相关的夹杂物。这些数据指出SGS和 TDP-43。该建议将解决SG依赖和独立过程在 ALS的病理生理学。我们假设SG生物学刺激了TDP-43包含物的形成， 与ALS相关的病原因素通过一个过程增加了TDP-43的纳入形成 由SG途径介导。 AIM 1将使用诱导的多能干细胞（IPSC，从 对照和TDP-43突变体细胞系）和海马神经元以表征调节 TDP-43包含形成。我们将使用成像来确定疾病连接的突变如何 TDP-43在基本或压力条件下修改RNA颗粒的形成和分散， 包括遗传毒性应激（例如，催化2的影响扩大的聚谷氨酰胺区域），兴奋性应激 （K+）或生长因子刺激。在每种情况下，我们都会评估RPC（包括SG）在 特定位置，例如soma或树突植物，通过将RPC形成限制为 细胞核，躯体或躯体/树突，并检查毒性。神经变性将由 在不同条件下测量树突长度以及树突结构的假定变化 将在人体组织中进行验证。在AIM 2中，我们将确定与TDP-43相关的分子因子 包含。我们将确定TDP-43或其他SG相关蛋白中的病理突变如何 修改条件下与TDP-43相关的蛋白质和mRNA。在目标3中我们 将确定TDP-43是否通过体内的SG增强机制形成包含。这个目标 将使用表达WT TDP-43的转基因小鼠将AIM 1和2的工作应用于体内设置。 我们将确定与夹杂物诱导的TDP-43 WT转基因小鼠相关的蛋白质。我们将 研究表达突变体TDP-43的检查转基因小鼠系，以确定是否是否 Ataxin-2 Q21、31或58的表达增加了TDP-43运动功能障碍，病理学。最后我们会的 确定ataxin-2敲除是否抑制TDP-43病理。调查特定元素 调节TDP-43纳入形成的SG途径将确定选择性的方法 治疗干预以延迟或停止ALS的进展。 呢