TDP-43, RNA Metabolism, and ALS/FTD Pathology

 描述（由申请人提供）：近年来在鉴定引起家族性肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）的基因方面取得了巨大进展，这两种疾病密切相关。这包括至少三种RNA结合蛋白（TDP-43、FUS和MATR 3）和C9 orf 72基因RNA转录物中的六核苷酸扩增。这些发现使人们更加关注RNA代谢的变化如何成为这些疾病的基础。虽然大多数ALS病例是散发性的（没有明显的家族遗传），但在几乎所有病例中，TDP-43蛋白都存在于受影响的运动神经元的细胞质内含物中，这表明该蛋白的功能与ALS广泛相关。我们在一个模型系统（C. elegans）和细胞培养，并发现这种蛋白质的缺失导致双链RNA（dsRNA）的积累和核糖体RNA的异常加工。本提案的目的是确定RNA代谢的这些变化是否在ALS/FTD病理学中发挥作用。我们将研究TDP-43限制dsRNA的分子机制，并试图确定FUS和MATR 3的缺失或C9 orf 72六核苷酸扩增的表达是否对RNA代谢具有类似的影响。还将研究TDP-43的疾病相关的细胞质再分布，特别是响应于C9 orf 72六核苷酸扩增的表达和易于聚集的聚二肽的相关产生。这些研究将采用RNA干扰、基因突变、免疫细胞化学、原位杂交和深度测序来全面表征RNA（转录组），使用人类细胞培养和C。elegans模型 我们将通过将这些研究扩展到患者细胞（成纤维细胞和重编程神经元）以及病理样本来测试我们的研究结果的疾病相关性。特别是，我们将测试我们已经确定的RNA变化可能在ALS病理学中涉及的神经炎症和星形胶质细胞功能障碍中发挥作用的假设。

 DESCRIPTION (provided by applicant): Dramatic advances have been made in recent years in the identification of genes that cause familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two closely related neurodegenerative diseases. This includes at least three RNA binding proteins (TDP-43, FUS and MATR3) and a hexanucleotide expansion in RNA transcripts of the C9orf72 gene. These findings have focused much attention on understanding how changes in RNA metabolism might underlie these diseases. Although most cases of ALS are sporadic (no obvious familial inheritance), in almost all cases the TDP-43 protein is found in cytoplasmic inclusions in affected motor neurons, suggesting the functions of this protein are broadly relevant to ALS. We have characterized the functions of TDP-43 in a model system (C. elegans) and cell culture, and discovered that loss of this protein results in accumulation of double-stranded RNA (dsRNA) and abnormal processing of ribosomal RNA. The goal of this proposal is to determine if these changes in the metabolism of RNA play a role in ALS/FTD pathology. We will investigate the molecular mechanisms by which TDP-43 limits dsRNA, and seek to determine if loss of FUS and MATR3, or expression of the C9orf72 hexanucleotide expansion, have similar effects on RNA metabolism. The disease-associated cytoplasmic redistribution of TDP-43 will also be investigated, particularly in response to expression of the C9orf72 hexanucleotide expansion and the associated production of aggregation-prone poly-dipeptides. These studies will employ RNA interference, genetic mutations, immunocytochemistry, in situ hybridization, and deep sequencing to globally characterize RNAs (the transcriptome), using both human cell culture and C. elegans models. We will test the disease relevance of our findings by extending these studies to patient cells (fibroblasts and reprogrammed neurons) as well as pathological samples. In particular, we will test the hypothesis that the RNA changes we have identified may play a role in the neuroinflammation and astroglial dysfunction that has been implicated in ALS pathology.