Investigating a Toxic Gain-of-Interaction Between FUS/TLS & Stress Granules

描述（由申请人提供）：最近与肌萎缩性侧索硬化症（ALS）有关的基因编码融合在肉瘤/易位在脂肪肉瘤（FUS/TLS或FUS）的突变。肌萎缩侧索硬化症是最常见的运动神经元疾病。平均发病年龄为55岁，确诊后患者一般仅存活3-5年。目前还没有治愈ALS的方法。大多数与als相关的FUS/TLS突变导致这种主要的核蛋白在细胞质内积累。迄今为止，尚不清楚这种错误定位是否通过诱导细胞核功能丧失和/或通过在细胞质中引入毒性功能的增加而在ALS发病机制中起作用。一些研究小组已经证明，als相关的突变FUS蛋白在对施加压力的反应中融入细胞质应激颗粒。相反，野生型FUS蛋白仍然是核的，并且在很大程度上被排除在应激颗粒之外。应激颗粒是一种停滞的翻译复合物，其功能是在应激诱导事件后恢复细胞稳态；应激颗粒是对应激的正常和必要的反应。我们的假设是，与ALS相关的突变FUS与应激颗粒的关联代表了一种毒性的相互作用增益，损害了这些颗粒的功能，从而损害了ALS患者的细胞应激反应和体内平衡。本提案的目的是确定突变fus与应力颗粒的关联是否会使用微观方法改变其功能特性（例如，这些应力颗粒在诱导应力下组装和拆卸的速率）。定量蛋白质组学将用于检查突变型FUS是否会改变细胞应激反应（即，在施加压力和应激消退期间细胞中的蛋白质翻译谱）。突变型fus掺入应激颗粒的机制将被研究，因为了解这一过程对未来的治疗目的很重要。利用肌萎缩性侧索硬化症（ALS）患者诱导的多能干细胞（iPS）和ALS转基因小鼠的运动神经元，研究突变fus相关应激颗粒对细胞稳态的影响。重要的是，上述这些研究将使我们能够解决突变的fus合并到应激颗粒中是否实际上是有毒的，从而可能导致疾病。FUS、应激颗粒和ALS发病机制之间的联系将通过探测人类患者样本中升高的应激颗粒标记物进一步研究。这些研究有可能揭示应激颗粒作为ALS的生物标志物。

DESCRIPTION (provided by applicant): Mutations in the gene encoding Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS or FUS) were recently linked to amyotrophic lateral sclerosis (ALS). ALS is the most common motor neuron disorder. The mean age of onset is 55 yrs, and patients generally survive for only 3-5 yrs after diagnosis. Currently there is no cure for ALS. A majority of ALS-linked mutations in FUS/TLS cause this predominately nuclear protein to accumulate within the cytoplasm. To date, it is not clear whether this mislocalization plays a role in ALS pathogenesis, either by inducing a loss of nuclear function and/or by introducing a gain of toxic function within the cytoplasm. Several groups have demonstrated that ALS-linked mutant FUS proteins incorporate into cytoplasmic stress granules in response to applied stress. Conversely, the wild-type FUS protein remains nuclear and largely excluded from stress granules. Stress granules are stalled translational complexes that function to restore cellular homeostasis after a stress-induced event; stress granules are a normal and necessary response to stress. Our hypothesis is that the association of ALS-linked mutant FUS with stress granules represents a toxic gain-of-interaction that impairs the function of these granules, thus compromising both cellular stress response and homeostasis in ALS. The aims of this proposal will determine if the association of mutant-FUS with stress granules alters their functional properties (e.g., rate at which these stress granules assemble and disassemble in response to induced stress) using microscopic methods. Quantitative proteomics will be used to examine whether mutant- FUS alters cellular stress response (i.e., the protein translation profile in the cell during applied stress and stress withdrawal). The mechanism of mutant-FUS incorporation into stress granules will be investigated, since it will be important to understand this process fo future therapeutic purposes. Motor neurons derived from ALS patient induced pluripotent stem (iPS) cells and ALS-transgenic mice will be employed to investigate the effects of mutant-FUS associated stress granules on cellular homeostasis. Importantly, these aforementioned studies will allow us to address whether mutant-FUS incorporation into stress granules is in fact toxic and thus potentially causative for disease. The link between FUS, stress granules and ALS pathogenesis will be further investigated by probing for elevated stress granule markers within human patient samples. These studies have the potential to reveal stress granules as biomarkers of ALS.