肌萎缩侧索硬化症（amyotrophic lateral sclerosis, ALS）是一种致死性的运动神经元病，目前尚无有效治疗方法。在已发现的50多个ALS致病基因中，有1/3参与RNA结合功能，如TARDBP和FUS，提示RNA代谢异常可能是ALS致病的关键因素。但是ALS具体的致病机制目前尚不清楚。合理的疾病动物模型对于疾病致病机制的研究至关重要，此前建立的能很好地还原ALS遗传特征的FUS-R521C突变敲入小鼠，没有出现严重的瘫痪和死亡，提示该小鼠存在遗传修饰。本论文以此突变敲入小鼠为基础，鉴定到了Trim72在FUS突变小鼠的神经系统中特异性上调表达。TRIM72（又称MG53），在骨骼肌和心脏中特异性高表达，参与保护多器官免受机械伤害、炎症和氧化应激等引起的损伤，但其是否在ALS中起保护作用以及其保护机制尚不清楚。我们发现Trim72的敲除可以加重FUS突变敲入小鼠的运动障碍、肌电异常以及运动神经元的死亡，确定了TRIM72参与FUS突变小鼠的疾病进展。进一步，我们发现Trim72的敲除会导致FUS突变小鼠脊髓中编码氧化磷酸化复合物相关的基因表达下调，升高FUS突变小鼠原代神经元的氧化应激水平，增加运动神经元中突变FUS蛋白的细胞质异常定位，上调应激颗粒标志蛋白的表达。由此揭示了Trim72的敲除使FUS突变小鼠出现典型ALS表型的原因是增加了氧化应激，为氧化应激和与ALS相关的致病突变的叠加在ALS疾病的发生发展的重要作用提供了证据。此外，我们发现TRIM72的抗氧化细胞保护作用不依赖于其E3连接酶活性，而与Coiled-coil和PRYSPRY结构域有关。TRIM72与CCC（COMMD/CCDC22/CCDC93）复合物相互作用，抗氧化作用受到参与内体货物分拣功能的CCDC22和retriever的调控，提示TRIM72的抗氧化作用可能与内体货物分拣机制有关。另外，在散发的ALS病人脑脊液中我们发现TRIM72的表达量与病人的疾病进程呈高度负相关。综上，我们的结果提示TRIM72在神经系统中特异性上调表达可能是解释FUS-R521C突变敲入小鼠未出现典型ALS表型的原因，TRIM72有望在治疗ALS等神经退行性疾病上发挥出潜在的价值。
Amyotrophic lateral sclerosis (ALS) is a fatal motor-neuron degenerative disease that is without effective treatment currently. Since about 1/3 of ALS causative genes are associated with RNA-binding, such as TARDBP and FUS, abnormal RNA metabolism has been recognized as one of the key pathogenesis in ALS. While the causes of ALS are currently not well understood. The FUS-R521C knock-in mouse established before can restore the genetic characteristics of ALS, and is free from severe motor defects and early death. Here, we identified a specific upregulation of Trim72 in the central nervous system of this mouse model. TRIM72, also called MG53, which is highly expressed in skeletal muscle and heart, is a key component of membrane repair and protects multiple organs from mechanical injury, oxidative stress and inflammation. Whether TRIM72 plays a protective role in ALS is currently unclear.We found that loss-of-function of Trim72 aggravates motor disability, abnormal electromyography, and motor neuron loss in FUS mutant mice. Furthermore, Trim72 deficiency results in down-regulation of genes encoding oxidative phosphorylation, elevated oxidative stress, increased cytoplasmic mislocalization of mutant FUS proteins and upregulation of stress granule marker proteins in FUS-R521C knock-in mice. Trim72 deficiency causes increased oxidative stress, therefore accelerating the disease progression of FUS-R521C knock-in mice. In addition, the antioxidant function of TRIM72 relies on the Coiled-coil and PRYSPRY domains, but not its E3 ligase activity. The antioxidant function of TRIM72 is also regulated by CCDC22 and retriever which are involved in endosomal cargo sorting. Besides, we found that the protein level of TRIM72 in the cerebrospinal fluid (CSF) is highly negatively correlated with the disease progression of ALS patients.Taken together, our results suggest that the upregulation of TRIM72 in the nervous system may underly the absence of typical ALS phenotypes in FUS-R521C knock-in mice. And TRIM72 is expected to have therapeutic potential in neurodegenerative diseases including ALS.
