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

描述（由申请方提供）：编码融合肉瘤/易位脂肪肉瘤（FUS/TLS或FUS）的基因突变最近与肌萎缩侧索硬化症（ALS）相关。ALS是最常见的运动神经元疾病。平均发病年龄为55岁，患者在诊断后通常只能存活3-5年。目前还没有治愈ALS的方法。 FUS/TLS中的大多数ALS连锁突变导致这种主要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核功能丧失和/或通过在细胞质内引入毒性功能的获得在ALS发病机制中起作用。几个研究小组已经证明，ALS连锁的突变FUS蛋白纳入细胞质应激颗粒响应于施加的压力。相反，野生型FUS蛋白保持核，并在很大程度上排除应力颗粒。应激颗粒是一种停滞的翻译复合物，其功能是在应激诱导的事件后恢复细胞内稳态;应激颗粒是对应激的正常和必要的反应。我们的假设是，ALS连锁突变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中的细胞应激反应和稳态。 该提案的目的将确定mu-FUS与应激颗粒的结合是否改变了它们的功能特性（例如，这些应力颗粒响应于诱导应力而组装和分解的速率）。定量蛋白质组学将用于检查突变型FUS是否改变细胞应激反应（即，在施加应力和应力消除期间细胞中的蛋白质翻译谱）。将研究mu-FUS掺入应激颗粒的机制，因为理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（iPS）细胞和ALS转基因小鼠的运动神经元将被用于研究与mu-FUS相关的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是否muplant-FUS掺入应激颗粒实际上是有毒的，从而潜在的致病性。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志物来进一步研究。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.