构建稳定表达TDP-43A315T突变的肌萎缩侧索硬化细胞模型并重现TDP-43蛋白病理特征的实验研究

Construction of a stable TDP-43A315T mutant cell model for amyotrophic lateral sclerosis recapitulating TDP-43 proteinopathy

摘要:

目的 通过在Neuro-2a细胞中构建稳定表达TDP-43^A315T的肌萎缩侧索硬化（ALS）体外模型，以期为药物筛选和致病机制的研究提供实验基础。方法 通过慢病毒介导的转染技术，在Neuro-2a细胞株中构建稳定表达野生型TDP-43与TDP-43^A315T突变型的细胞株。该研究设置3个分组，分别为空载组（GFP-Control组，简称GL组）转染质粒pSLenti-EF1-EGFP-P2A-Puro-CMVMCS-3xFLAG-WPRE；对照组（野生型人类TDP-43序列，简称WT组）转染质粒pSLenti-EF1-EGFP-P2A-Puro-CMV-TARDBP-3xFLAG-WPRE；模型组（人类TDP-43A315T突变序列，简称M组）转染质粒pSLenti-EF1-EGFP-P2A-Puro-CMV-TARDBP（A315T）-3xFLAG-WPRE。采用CCK-8法和腺苷三磷酸（ATP）含量检测评估细胞活力与能量代谢；通过Western blotting检测和免疫荧光染色实验分析TDP-43的亚细胞定位、剪切片段形成、泛素化聚集及凋亡相关蛋白的表达；利用生化试剂盒检测氧化应激相关酶活性变化。结果 GL组、WT组ATP蛋白相对表达量均高于M组（P <0.05）。M组的cleaved Caspase-3、B淋巴细胞瘤-2基因相关启动子、Bcl-2相关X蛋白的表达量均高于GL组及WT组（P <0.05）。M组TDP-43免疫荧光染色在细胞质中的占比高于WT组（P <0.05）。M组细胞质TDP-43蛋白水平高于WT组（P <0.05）。M组5、15、20 μmol/L过氧化氢干预后细胞存活率均高于GL组、WT组（P <0.05），40、50 μmol/L过氧化氢干预后细胞存活率均低于GL组、WT组（P <0.05）。M组T-AOC、T-GSH均高于GL组、WT组（P <0.05）。M组Nrf2、HO-1表达水平均低于WT组、GL组（P <0.05）。结论 该研究成功构建了1个稳定表达和典型病理特征的TDP-43^A315TNeuro-2a细胞模型。TDP-43^A315T突变通过诱导线粒体功能障碍、细胞凋亡、TDP-43蛋白病理改变及氧化应激防御系统失调，协同导致神经元损伤，为解析ALS发病机制及药物筛选提供了体外研究工具。

Abstract:

Objective The core pathology of amyotrophic lateral sclerosis (ALS) involves cytoplasmic mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43) coupled with its nuclear clearance. The TDP-43^A315T mutation is a well-established pathogenic mutation. This study aimed to construct a stable in vitro ALS model by expressing human TDP-43^A315T in Neuro-2a cells to facilitate new drug development and genetic screening.Methods Lentivirus-mediated transduction was used to establish stable Neuro-2a cell lines expressing either wild-type (WT) or A315T mutant human TDP-43 (hTDP-43). The study established three groups: the empty vector group (GFP-Control group, abbreviated as GL group) transfected with the plasmid pSLenti-EF1-EGFP-P2A-Puro-CMV-MCS-3xFLAG-WPRE; the control group (wild-type human TDP-43 sequence, abbreviated as WT group) transfected with the plasmid pSLenti-EF1-EGFP-P2A-Puro-CMV-TARDBP-3xFLAG-WPRE; and the model group (human TDP-43 A315T mutant sequence, abbreviated as M group) transfected with the plasmid pSLenti-EF1-EGFP-P2A-Puro-CMV-TARDBP (A315T)-3xFLAG-WPRE. Cell viability and energy metabolism were assessed using CCK-8 assay and ATP level measurement. The subcellular localization of TDP-43, formation of cleaved fragments, ubiquitinated aggregates, and expression of apoptosis-related proteins were analyzed by Western Blotting and immunofluorescence staining. Activities of key enzymes related to oxidative stress were measured using biochemical assay kits.Results The relative expression of ATP protein in the GL and WT groups was higher than that in the M group (P < 0.05). The expression levels of cleaved Caspase-3, BAD, and BAX in the M group were higher than those in the GL and WT groups (P < 0.05). The proportion of cytoplasmic TDP-43 immunofluorescence staining in the M group was higher than that in the WT group (P < 0.05), and the cytoplasmic TDP-43 protein level was also higher in the M group than in the WT group (P < 0.05). After exposure to 5, 15, and 20 μmol/L hydrogen peroxide (H₂O₂), the cell viability in the M group was higher than that in the GL and WT groups (P < 0.05), whereas after 40 and 50 μmol/L H₂O₂ exposure, cell viability in the M group was lower than that in the GL and WT groups (P < 0.05). The levels of total antioxidant capacity (T-AOC) and total glutathione (T-GSH) in the M group were higher than those in the GL and WT groups (P < 0.05). In contrast, the expression levels of Nrf2 and HO-1 in the M group were lower than those in the WT and GL groups (P < 0.05).Conclusion A stable TDP-43^A315T Neuro-2a cell model with typical pathological features was successfully established. The TDP-43^A315T mutation induces mitochondrial dysfunction, apoptosis, pathological TDP-43 protein alterations, and dysregulation of the oxidative stress defense system, which collectively contribute to neuronal injury, providing an in vitro model for elucidating the pathogenesis of ALS and for drug screening.