Cellular and Animal Models of FUS Mutations in ALS

描述（由申请人提供）：本R21申请是根据PA-10-138“研究用动物模型和相关生物材料的开发”编写的。在本申请中，我们建议建立细胞和动物模型，使我们能够表征FUS/TLS突变的机制。人类16号染色体上的FUS/TLS基因座最初被鉴定为编码与恶性脂肪肉瘤和髓性白血病有关的癌基因。最近的证据表明，FUS/TLS突变发现于肌萎缩侧索硬化症（ALS）的家族性病例中。这些结果表明，进一步 对FUS/TLS基因作用机制的研究将对肿瘤生物学、神经退行性变、衰老和精神健康等生物医学研究领域产生深远的影响。虽然大多数突变被发现在高度保守的C-末端区域的FUS蛋白，这些突变的确切致病机制尚未完全阐明。在FUS突变中，成人ALS患者中最常见的形式是R521 C突变。此外，我们最近的研究表明，FUS中的P525 L突变导致年轻患者中ALS的更具侵袭性和快速进展形式。目前，尚不清楚FUS-R521 C和FUS-P525 L如何导致神经元变性，或者为什么FUS-P525 L倾向于影响年轻患者。然而，我们最近的数据表明，P525 L突变患者的脊髓运动神经元显示突变FUS蛋白在神经元细胞质中的错误定位，异常FUS蛋白聚集体与明显紊乱的细胞内细胞器，包括内质网（ER）和线粒体相关。这些结果使我们假设突变FUS蛋白主要抑制正常RNA和蛋白质合成，从而抑制神经元功能和存活。我们的目标是进一步研究突变FUS蛋白在神经元变性的机制，使用原代神经元和转基因小鼠。由于突触变性在神经退行性疾病中起着核心作用，我们相信这些模型的建立将有助于理解突变FUS蛋白在脊髓内和神经肌肉接头（NMJ）的运动神经元的突触连接的发展和维持。我们提出两个具体目标来实现这些目标。在目的1中，我们将描述FUS-R521 C和FUS-P525 L突变蛋白影响原代神经元突触变性和细胞死亡的机制。在目的2中，我们提出表征表达FUS-R521 C或FUS-P525 L的小鼠是否发展与具有相应突变的患者相似的运动行为表型和运动神经元病理学。我们相信，这些模式的建立将对NIH许多机构的使命做出重要贡献，并满足R21资助机制“高回报和高影响”的标准。一旦可用，这些模型将作为新的平台，为FUS/TLS突变引起的疾病确定新的治疗靶点。

DESCRIPTION (provided by applicant): This R21 application is prepared in response to PA-10-138, "Development of Animal Models and Related Biological Materials For Research". In this application, we propose to establish both cellular and animal models that will allow us to characterize the mechanism of FUS/TLS mutations. The FUS/TLS locus on human chromosome 16 is originally identified to encode an oncogene implicated in malignant liposarcoma and myeloid leukemia. More recent evidence shows that mutations in FUS/TLS are found in familial cases of amyotrophic lateral sclerosis (ALS). These results indicate that further investigations to the mechanisms of FUS/TLS gene will have profound impacts on many disciplines in biomedical research, including cancer biology, neurodegeneration, aging, and mental health. Although most mutations are found in the highly conserved C-terminal region of the FUS protein, the precise pathogenic mechanisms for these mutations have not been fully elucidated. Among the FUS mutations, the most common form in adult ALS patients is the R521C mutation. In addition, our recent study indicates that the P525L mutation in FUS leads to a more aggressive and rapidly progressive form of ALS in young patients. Currently, it is unclear how FUS-R521C and FUS-P525L cause neuronal degeneration, or why FUS-P525L tends to affect younger patients. Our recent data, however, indicated that the spinal motor neurons in patients with P525L mutation show mislocalization of mutant FUS proteins in neuronal cytoplasm with abnormal FUS protein aggregates that are associated with markedly disorganized intracellular organelles, including endoplasmic reticulum (ER) and mitochondria. These results lead us to hypothesize that mutant FUS proteins dominantly inhibit normal RNA and protein synthesis and thereby suppressing neuronal functions and survival. Our objectives are to further investigate the mechanisms of mutant FUS proteins in neuronal degeneration using both primary neurons and transgenic mice. Since synaptic degeneration plays a central role in neurodegenerative diseases, we believe that the establishment of these models will contribute to understanding of the mutant FUS proteins in the development and maintenance of synaptic connectivity of motor neurons within the spinal cord and at the neuromuscular junction (NMJ). We propose two specific aims to achieve these goals. In Aim 1, we will characterize the mechanisms by which FUS-R521C and FUS-P525L mutant proteins affect synaptic degeneration and cell death in primary neurons. In Aim 2, we propose to characterize if mice expressing FUS-R521C or FUS-P525L develop motor behavioral phenotype and motor neuron pathology similar to patients with corresponding mutations. We believe that the establishment of these models will have important contributions to the missions of many institutions at NIH, and fulfills the criteria of "high rewards and high impacts" for the R21 funding mechanism. Once available, these models will serve as novel platforms to identify new therapeutic targets for diseases caused by FUS/TLS mutations.