FUS-R521C基因敲入ALS小鼠模型中潜在治疗靶标的探究

在30多个肌萎缩侧索硬化（ALS）致病基因中，有1/3编码RNA结合蛋白（RBP），包括TDP-43和FUS，但是RBP功能异常如何致病的机理不清。既往研究提示RBP参与应激颗粒（stress granule, SG）的代谢。SG具有相分离特性，其形成抑制胞质mRNA翻译，在胁迫解除后SG解聚，帮助细胞应对胁迫应激。通过突变敲入的方式，我构建了FUS突变敲入的ALS小鼠模型，利用双光子成像技术，我跟踪到SG在突变小鼠皮层异常加工，提示突变的RBP通过影响SG的相分离特性而致病。在突变小鼠的神经组织和脑脊液中，我发现一个E3连接酶Trim72有约十倍的上调。Trim72的功能缺失加重突变小鼠表型和SG的代谢紊乱。本项目拟解决Trim72是否通过抗氧化缓解SG的代谢紊乱进而实现其神经保护作用，以及ALS病人脑脊液Trim72含量是否与疾病进程相关。我们希望本研究为未来疾病的诊治提供潜在靶点。

Mutations in and dysfunction of genes encoding RNA-binding proteins (RBPs), including TDP-43 and FUS, have been associated with amyotrophic lateral sclerosis (ALS). These RBPs often contain RNA recognition motif and low complexity domain and are found in a membrane-less cellular structure, also known as stress granule (SG), an RNA/protein complex that appears under stress to temporarily cease mRNA translation initiation. In the past, significant efforts have been made to generate animal models for ALS by overexpressing recombinant DNA carrying mutations found in ALS families, including TDP-43 and FUS. Although those transgenic animal models significantly gain our insights into the disease mechanisms, people may argue the potential artifacts driven by ectopic overexpression of mutant proteins. To avoid the caveats of transgenic approach, we previously took knock-in (KI) approach to generate FUS-R521C KI ALS mouse model and documented motor ability decline and motor neuron loss in this KI animal (PMID: 32358598). In addition, we traced SG misprocessing in the mutant motor cortex before and after stress challenge by two-photon imaging and correlated with the severity of SG misprocessing with neuron fate. In the FUS-R521C KI mouse neuronal tissues and cerebrospinal fluid (CSF), we found ~10-time upregulation of Trim72 an E3 ligase. Loss-of-function of Trim72 induces oxidative stress and SG protein upregulation in the mutant neuron and brain. In this proposal, we propose to express Trim72 in the mutant neuron and motor cortex and examine its neuronal protective effect against oxidative stress and SG misprocessing. In addition, we plan to correlate the CSF TRIM72 expression level with disease progression of ALS. Therefore, identification of Trim72 from a KI ALS mouse model we generated as a potential therapeutic target may benefit ALS patients and CSF TRIM72 level may predict prognosis of ALS.