Toxic gain of function from mutant FUS protein is crucial to trigger cell autonomous motor neuron loss

FUS is an RNA-binding protein involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cytoplasmic FUS-containing aggregates are often associated with concomitant loss of nuclear FUS. Whether loss of nuclear FUS function, gain of a cytoplasmic function, or a combination of both lead to neurodegeneration remains elusive. To address this question, we generated knock-in mice expressing mislocalized cytoplasmic FUS and complete FUS knock-out mice. Both mouse models display similar perinatal lethality with respiratory insufficiency, reduced body weight and length, and largely similar alterations in gene expression and mRNA splicing patterns, indicating that mislocalized FUS results in loss of its normal function. However, FUS knock-in mice, but not FUS knock-out mice, display reduced motor neuron numbers at birth, associated with enhanced motor neuron apoptosis, which can be rescued by cell-specific CRE-mediated expression of wild-type FUS within motor neurons. Together, our findings indicate that cytoplasmic FUS mislocalization not only leads to nuclear loss of function, but also triggers motor neuron death through a toxic gain of function within motor neurons. Total RNA from brains of E18.5 knock-in (FusdeltaNLS/deltaNLS), and knock-out (Fus-/-) mice and their control littermates were extracted with Trizol and libraries were prepared for RNA sequencing and RNA-mediated oligonucleotide Annealing, Selection, and Ligation with Next-Generation sequencing (RASL-seq) (Li et al. 2012; Zhou et al. 2012). For the RNA-seq experiment, biological replicates were used with n=4-5 knock-in animals per group (4 wild-type and 5 homozygous FusdeltaNLS) and n=5 knock-out animals per group (5 wild-type and 5 homozygous Fus-/-). For the RASL-seq analysis, biological replicates were used with n=4 knock-in animals per group (4 wild-type, 4 heterozygous and 4 homozygous FusdeltaNLS) and n=5 animals per group (5 wild-type and 5 homozygous Fus-/- knock-out).FUS蛋白是一种RNA结合蛋白，与肌萎缩侧索硬化症（amyotrophic lateral sclerosis, ALS）和额颞叶痴呆（frontotemporal dementia, FTD）密切相关。细胞质中含FUS的聚集物常伴随细胞核内FUS的表达缺失。目前尚不清楚究竟是核内FUS功能缺失、胞质内FUS的毒性功能获得，还是两者共同作用导致了神经退行性病变。为厘清这一科学问题，我们构建了表达胞质错位定位FUS的基因敲入小鼠，以及完全敲除FUS的基因敲除小鼠。两种小鼠模型均表现出相似的围产期致死表型，伴随呼吸功能不全、体重与体长降低，且基因表达和mRNA剪接模式整体高度相似，表明错位定位的FUS会导致其正常功能丧失。但仅FUS基因敲入小鼠（而非基因敲除小鼠）在出生时出现运动神经元数量减少，并伴随运动神经元凋亡水平升高，该表型可通过在运动神经元中细胞特异性表达野生型FUS的Cre重组酶介导系统实现挽救。综上，本研究结果表明，胞质FUS错位定位不仅会引发核内功能缺失，还会通过在运动神经元内获得毒性功能，触发运动神经元死亡。我们采用Trizol试剂提取了E18.5胎龄的FUS基因敲入（FusΔNLS/ΔNLS）、FUS基因敲除（Fus-/-）小鼠及其同窝对照小鼠的脑组织总RNA，并构建文库用于RNA测序（RNA-seq）以及RNA介导的寡核苷酸退火、选择与连接测序（RNA-mediated oligonucleotide Annealing, Selection, and Ligation with Next-Generation sequencing, RASL-seq）（Li等，2012；Zhou等，2012）。在RNA-seq实验中，设置生物学重复：每组敲入小鼠n=4~5（4只野生型、5只纯合型FusΔNLS/ΔNLS小鼠），每组敲除小鼠n=5（5只野生型、5只纯合型Fus-/-小鼠）。在RASL-seq分析中，同样设置生物学重复：每组敲入小鼠n=4（4只野生型、4只杂合型、4只纯合型FusΔNLS/ΔNLS小鼠），每组敲除小鼠n=5（5只野生型、5只纯合型Fus-/-小鼠）。