TRAP-seq of spinal cord motor neurons from ALS diseased mice versus healthy mice

Amyotrophic lateral sclerosis (ALS) is an incurable neurological disease featuring progressive loss of motor neuron (MN) function in the brain and spinal cord. Mutations in TARDBP, encoding the RNA-binding protein TDP-43, are one cause of ALS and TDP-43 mislocalization in MNs is a key pathological feature of >95% of ALS cases. While numerous studies support altered RNA regulation by TDP-43 as a major cause of disease, specific changes within MNs that trigger disease onset remain unclear. Here, we combined Translating Ribosome Affinity Purification (TRAP) with RNA sequencing to identify molecular changes in spinal MNs of TDP-43–driven ALS at motor symptom onset. By comparing the MN translatome of hTDP-43A315T mice to littermate controls and to mice expressing wildtype hTDP-43, we identify hundreds of mRNAs that were selectively up- or downregulated in MNs. We validated effects on Tex26, Syngr4, and Plekhb1 mRNAs in an independent TRAP experiment. Moreover, by quantitative immunostaining of spinal cord MNs we found corresponding protein level changes for SYNGR4 and PLEKHB1. We also observed these changes in spinal MNs of an independent ALS mouse model caused by a different patient mutant allele of TDP-43, suggesting that they may be a general feature of TDP-43-driven ALS. Thus, we have identified two new proteins deregulated in MNs at motor symptom onset in TDP-43-driven ALS models. This spatial and temporal pattern suggests that deregulation of these proteins could be functionally important for driving the transition to the symptomatic phase of disease. Overall design: mRNA of Chat+ spinal cord motor neurons derived from mice with TDP43^A315T mutation and control mice (wildtype and TDP43^WT) was enriched by translating ribosome affinity purification (TRAP) and sequenced by RNA-seq.

肌萎缩侧索硬化症（Amyotrophic lateral sclerosis, ALS）是一种无法治愈的神经系统疾病，以脑与脊髓内运动神经元（motor neuron, MN）功能进行性丧失为特征。编码RNA结合蛋白TDP-43（RNA-binding protein TDP-43）的TARDBP基因突变是ALS的致病诱因之一，而运动神经元内TDP-43的异常定位是超过95% ALS病例的关键病理特征。尽管大量研究证实TDP-43介导的RNA调控异常是疾病发生的主要诱因，但触发疾病发作的运动神经元内特异性分子变化仍未明确。
本研究将翻译核糖体亲和纯化（Translating Ribosome Affinity Purification, TRAP）技术与RNA测序相结合，以鉴定运动症状发作期TDP-43驱动型ALS小鼠脊髓运动神经元的分子改变。通过对比hTDP-43A315T小鼠与同窝对照小鼠、以及表达野生型hTDP-43小鼠的运动神经元翻译组，我们筛选得到数百个在运动神经元中发生选择性上调或下调的信使RNA（mRNA）。
我们在独立的TRAP实验中验证了Tex26、Syngr4及Plekhb1 mRNA的表达变化。此外，通过对脊髓运动神经元进行定量免疫染色，我们发现SYNGR4与PLEKHB1的蛋白水平也出现了相应改变。我们还在另一种由不同TDP-43患者突变等位基因构建的ALS小鼠模型的脊髓运动神经元中观察到了上述变化，提示这些变化可能是TDP-43驱动型ALS的共性特征。
综上，我们鉴定出两种在TDP-43驱动型ALS模型运动症状发作期于运动神经元内表达失调的新型蛋白。这种时空表达模式表明，这些蛋白的表达失调可能在疾病向症状期进展的过程中发挥关键的功能作用。
实验设计：本研究通过翻译核糖体亲和纯化（TRAP）技术富集了携带TDP43^A315T突变小鼠及对照小鼠（野生型及TDP43^WT）来源的Chat+脊髓运动神经元的mRNA，并通过RNA-seq完成测序分析。