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）中内含物的主要成分，并且在一些实施例中，TDP-43是肌萎缩性侧索硬化症（ALS）中内含物的主要成分。 额颞叶痴呆（FTLD-U）。TDP-43是核RNA结合蛋白，其易位至 细胞质在压力下形成细胞质颗粒。我们的研究表明， 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 模型和人类大脑中。TDP-43中的疾病相关突变也增加了 与SG相关的夹杂物。这些数据表明，SG和 TDP-43本提案将讨论依赖于秘书长和独立于秘书长的进程在 ALS的病理生理学我们假设SG生物学刺激TDP-43内含物的形成， 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， 由SG通路介导。目的1将使用诱导多能干细胞（IPSC，由 对照和TDP-43突变体人细胞系）和海马神经元以表征调节 TDP-43包合物形成。我们将使用成像来确定疾病相关的突变是如何在 TDP-43在基础或应激条件下改变RNA颗粒的形成和分散， 包括遗传毒性应激（例如，共济失调蛋白-2的作用（扩大的多聚谷氨酰胺区域），兴奋性应激 （K+）或生长因子刺激。在每种情况下，我们评估RPC（包括SG）在以下方面的作用： 特定的位置，如索马或树枝状乔木，通过遗传限制RPC的形成， 细胞核、索马或索马/树突，并检测毒性。将通过以下方法监测神经变性： 测量不同条件下的树枝状长度，以及树枝状结构的推定变化 将在人体组织中进行验证。在目标2中，我们将确定与TDP-43相关的分子因子 内含物。我们将确定TDP-43或其他SG相关蛋白的病理性突变 修饰的蛋白质和mRNA与TDP-43条件下的夹杂物。在目标3中， 将确定TDP-43是否通过SG增强的体内机制形成包合物。这一目标 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。 我们将在诱导型TDP-43 WT转基因小鼠中鉴定与包涵体相关的蛋白质。我们将 研究检测表达突变TDP-43的转基因小鼠系，以确定是否 共济失调蛋白-2 Q21、31或58的表达增加TDP-43运动功能障碍，病理学。最后我们将 确定共济失调蛋白-2敲除是否抑制TDP-43病理。调查特定元素 调节TDP-43包涵体形成的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。 !