Mutant Human FUS Is Ubiquitously Mislocalized and Generates Persistent Stress Granules in Primary Cultured Transgenic Zebrafish Cells

FUS mutations can occur in familial amyotrophic lateral sclerosis (fALS), a neurodegenerative disease with cytoplasmic FUS inclusion bodies in motor neurons. To investigate FUS pathology, we generated transgenic zebrafish expressing GFP-tagged wild-type or fALS (R521C) human FUS. Cell cultures were made from these zebrafish and the subcellular localization of human FUS and the generation of stress granule (SG) inclusions examined in different cell types, including differentiated motor neurons. We demonstrate that mutant FUS is mislocalized from the nucleus to the cytosol to a similar extent in motor neurons and all other cell types. Both wild-type and R521C FUS localized to SGs in zebrafish cells, demonstrating an intrinsic ability of human FUS to accumulate in SGs irrespective of the presence of disease-associated mutations or specific cell type. However, elevation in relative cytosolic to nuclear FUS by the R521C mutation led to a significant increase in SG assembly and persistence within a sub population of vulnerable cells, although these cells were not selectively motor neurons.

FUS突变可发生于家族性肌萎缩侧索硬化症（familial amyotrophic lateral sclerosis，fALS），该病是一类以运动神经元胞质内存在FUS包涵体的神经退行性疾病。为探究FUS相关病理机制，我们构建了表达绿色荧光蛋白（GFP）标记的野生型或携带R521C突变的人源FUS的转基因斑马鱼。我们从这些转基因斑马鱼中制备了细胞培养物，并检测了人源FUS的亚细胞定位，以及不同细胞类型（包括分化的运动神经元）中应激颗粒（stress granule，SG）包涵体的生成情况。
我们证实，突变型FUS在运动神经元及所有其他细胞类型中，均以相似程度从细胞核异常移位至胞质。在斑马鱼细胞中，野生型与R521C突变型FUS均定位于应激颗粒，这表明人源FUS具有内在的在应激颗粒中聚集的能力，且不受疾病相关突变或特定细胞类型的影响。
然而，R521C突变使胞质FUS相对于核FUS的比例升高，会导致应激颗粒组装显著增加，并在部分易感细胞亚群中持续存在，尽管这些细胞并非特异性的运动神经元。