Stress Granules and the Biology of TDP-43

描述(申请人提供)：TDP-43是肌萎缩侧索硬化症(ALS)和一些额颞叶痴呆(FTLD-U)中包涵体的主要成分。TDP-43是一种核RNA结合蛋白，在应激过程中移位到细胞质，在那里形成细胞质颗粒。我们的研究表明，在细胞模型和人脑中，这些细胞质TDP-43包涵体与被称为“应激颗粒”(SGS)的RNA颗粒共存。TDP-43的疾病相关突变也增加了与SGS相关的包涵体的形成。这些数据表明SGS和TDP-43之间有很强的生物学联系。这项建议将阐述SG依赖和独立过程在ALS病理生理学中的作用。我们假设SG生物学刺激TDP-43包涵体的形成，与ALS相关的致病因素通过SG通路介导的过程增加TDP-43包涵体的形成。目的1利用诱导多能干细胞(IPSCs)和海马神经元研究TDP-43包涵体形成的调控。我们将使用成像来确定TDP-43中与疾病相关的突变如何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，ataxin-2？多聚谷氨酰胺区域扩大)、兴奋性应激(K+)或生长因子刺激。在每种情况下，我们通过从基因上将RPC的形成限制在细胞核、胞体或胞体/树突并检测毒性，来评估RPC(包括SGS)在特定位置的作用，例如胞体或树突。神经退行性变将通过测量不同条件下的树突长度来监测，树突结构的假定变化将在人体组织中得到验证。在目标2中，我们将确定与TDP-43包涵体相关的分子因素。我们将确定TDP-43或其他SG相关蛋白的病理性突变如何在条件下改变与TDP-43相关的蛋白质和mRNA？包裹体。在目标3中，我们将确定TDP-43是否在体内通过SG增强的机制形成包合物。这一目标将把AIMS 1和AIMS 2的工作应用于体内环境，使用表达WT TDP-43的转基因小鼠。我们将在可诱导的TDP-43WT转基因小鼠中鉴定与包涵体相关的蛋白质。我们将研究表达突变型TDP-43的转基因小鼠系，以确定ataxin-2Q21、31或58的表达是否会增加TDP-43的运动功能障碍和病理学。最后，我们将确定ataxin-2基因敲除是否抑制TDP-43的病理。研究调节TDP-43包涵体形成的SG通路的特定元件将确定选择性的治疗干预方法，以延缓或抑制ALS的进展。 与公共卫生相关：TDP-43是一种与RNA包涵体相关的RNA结合蛋白，称为应激颗粒。这项建议将集中在RNA蛋白复合体和应激颗粒途径，以确定与TDP-43包涵体相关的分子因素，并确定这些因素如何调节与TDP-43相关的神经退行性变。

DESCRIPTION (provided by applicant): 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 hal the progression of ALS. PUBLIC HEALTH RELEVANCE: TDP-43 is a RNA binding protein that associates with RNA inclusions, termed stress granules. This proposal will focus on RNA protein complexes and the stress granule pathway to identify molecular factors associated with TDP-43 inclusions and determine how these factors regulate neurodegeneration related to TDP-43.