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

描述(申请人提供)：编码肉瘤融合/脂肪肉瘤转位的基因突变(FUS/TLS或FUS)最近被认为与肌萎缩侧索硬化症(ALS)有关。肌萎缩侧索硬化是最常见的运动神经元疾病。平均发病年龄为55岁，患者一般在确诊后仅存活3-5年。目前还没有治愈肌萎缩侧索硬化症的方法。大多数与ALS相关的FUS/TLS突变都会导致这种以核蛋白为主的蛋白在细胞质内积聚。到目前为止，这种定位错误是否在ALS的发病机制中发挥作用尚不清楚，无论是通过导致核功能丧失和/或通过在细胞质内引入毒性功能。几个小组已经证明，ALS连锁突变的FUS蛋白结合到细胞质应激颗粒中，以响应施加的应激。相反，野生型FUS蛋白仍然是核的，在很大程度上被排除在应激颗粒之外。应激颗粒是停滞的翻译复合体，其功能是在应激诱导事件后恢复细胞动态平衡；应激颗粒是对应激的正常和必要的反应。我们的假设是，ALS连锁突变FUS与应激颗粒的关联代表了一种有毒的相互作用获得，损害了这些颗粒的功能，从而损害了ALS的细胞应激反应和内稳态。这项提议的目的将使用微观方法确定突变-FU与应激颗粒的关联是否改变了它们的功能属性(例如，这些应激颗粒对诱导应激的组装和拆解速度)。定量蛋白质组学将被用来检查突变的FUS是否改变了细胞的应激反应(即，在施加应激和应激退出期间细胞中的蛋白质翻译谱)。我们将研究突变体-FUS整合到应激颗粒中的机制，因为了解这一过程对于未来的治疗目的将是重要的。来自ALS患者诱导的多能干细胞和ALS转基因小鼠的运动神经元将被用来研究突变-FUS相关应激颗粒对细胞内稳态的影响。重要的是，上述研究将使我们能够解决突变-FUS整合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在人类患者样本中探测升高的应激颗粒标记物来进一步研究。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.