TDP-43 Interactor and Motor Neuron Tagged Ribosome Affinity Purification in a Drosophila Model of ALS

We report the enrichment of mRNAs with motor neuron specific TDP-43 and tagged ribosomes in control as well as multiple models of TDP-43 induced neurodegeneration. Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative disease in which 97% of patients exhibit cytoplasmic aggregates containing the RNA binding protein TDP-43. The goal of this study is to understand the translational consequences of TDP-43 pathology. Using the GAL4-UAS system, we expressed TDP-43WT and TDP-43G298S in the motor neurons of our drosophila to induce ALS-like neurodegeneration. TDP-43 was immunoprecipitated from the larvae at the 3rd instar stage; TDP-43 associated mRNAs, the whole larvae input, and ventral nerve cords of the same genotype were then sequenced. To identify translational changes we conducted TRAP (translating ribosome affinity purifications) in 3rd instar larvae by immunoprecipitating the motor neuron specific tagged ribosomal subunit RpL10-GFP in both ALS models as well as a RpL10-GFP control. The RpL10-GFP associated mRNAs, the whole larval input, and ventral nerve cords of the same genotype. From this data, we identified both compensatory alterations to translation induced by TDP-43 pathology and direct targets of TDP-43 mediated translational inhibition. Examination of mRNA association with TDP-43 and tagged ribosomal subunit RpL10 in motor neurons during TDP-43 induced neurodegeneration. The antibody used for both IPs was rabbit anti-GFP Lifetech A11122.

本研究报道了对照组及多种TDP-43诱导神经退行性变模型中，结合运动神经元特异性TDP-43与标记核糖体的mRNA的富集特征。肌萎缩侧索硬化症（Amyotrophic lateral sclerosis, ALS）是一种遗传异质性神经退行性疾病，97%的患者体内会出现包含RNA结合蛋白TDP-43的细胞质聚集物。本研究的目标是阐明TDP-43病理改变所带来的翻译调控后果。本研究借助GAL4-UAS系统，在果蝇的运动神经元中表达野生型TDP-43（TDP-43WT）与突变型TDP-43G298S，以诱导出类似ALS的神经退行性表型。研究人员于三龄幼虫阶段提取样本，对TDP-43进行免疫沉淀（immunoprecipitation, IP）；随后对与TDP-43结合的mRNA、同基因型的全幼虫输入对照样本以及腹神经索进行测序。为了鉴定翻译水平的变化，本研究在三龄幼虫中开展了TRAP（翻译核糖体亲和纯化，translating ribosome affinity purifications）实验：通过免疫沉淀结合运动神经元特异性标记核糖体亚基RpL10-GFP，分别在ALS模型组与RpL10-GFP对照组中完成该操作。随后对与RpL10-GFP结合的mRNA、同基因型的全幼虫输入对照样本以及腹神经索进行测序。基于上述实验数据，本研究既鉴定出了TDP-43病理诱导的翻译代偿性改变，也找到了TDP-43介导的翻译抑制的直接靶标。本研究同时分析了TDP-43诱导神经退行性变过程中，运动神经元内mRNA与TDP-43及标记核糖体亚基RpL10的结合情况。两次免疫沉淀实验所使用的抗体均为兔抗GFP抗体（Lifetech A11122）。