Identification of pathogenic domain in TDP-43 for ALS toward the antibody medicine

通过对als连锁蛋白TDP-43的rna识别基序（RRM）的结构分析，我们在RRM1中发现了一个异常的组装界面。该区域的半胱氨酸诱导TDP-43聚集、细胞质错定位、RNA剪接不正常和细胞毒性，在功能和结构上模仿TDP-43蛋白病变。我们还成功克隆了小鼠杂交瘤（3B12A）的cDNA，生成了识别TDP-43错误折叠形式的单克隆抗体，并构建了单链Fv （scFv）的哺乳动物表达质粒。由于市场上可获得的表达ALS相关突变体TDP-43 （A315T）的转基因小鼠无法复制ALS表型，我们利用子宫内电穿孔技术建立了胚胎脑转染系统。表达聚集易感突变体TDP-43的脑切片显示als样TDP-43聚集，可用于评估体内治疗。

Using structural analyses of RNA-recognition motifs (RRM) of TDP-43, a ALS-linked protein, we identified a aberrant assembly interface in RRM1. The cysteine in the this region induced TDP-43 aggregates, cytosolic mislocalization, the incompetent RNA splicing, and cytotoxicity, which functionally and structurally mimicks TDP-43 proteinopathy. We also succeeded in cloning cDNA from murine hybridoma (3B12A), generating a monoclonal antibody recognizing the misofolded forms of TDP-43, and constructed mammalian expression plasmid for the single chain Fv (scFv). Since the commercially available transgenic mice expressing ALS-linked mutant TDP-43 (A315T) turned out to fail to replicate ALS phenotype, we newly establish the embryonic brain transfection system, using in-utero electroporation. The brain slice expressing aggregate prone mutant TDP-43 showed ALS-like TDP-43 aggregates, which could be tested to estimate the intrabody therapy.