Role of NTRK2 on ReNcelVM differentiation

The tropomyosin receptor kinase B (TrkB) is encoded by the NTRK2 gene. It belongs to the family of transmembrane tyrosine kinases, which have key roles in the development and maintenance of the nervous system. Brain-derived neurotrophic factor (BDNF) and the neurotrophins NT3 and NT4/5 have high affinity for TrkB. Dysregulation of TrkB is associated to a large spectrum of diseases including neurodegeneration, psychiatric diseases and some cancers. The function of TrkB and its role in neural development have mainly been decrypted using transgenic mouse models, pharmacological modulators and human neuronal cell lines overexpressing NTRK2. In this study, we identified high expression and robust activity of TrkB in ReNcell VM, an immortalized human neural progenitor stem cell line and generated NTRK2-deficient (NTRK2-/-) ReNcell VM using the CRISPR/Cas9 gene editing technology. Global transcriptomic analysis revealed major changes in expression of specific genes responsible for neurogenesis, neuronal development and glial differentiation. In particular, key neurogenic transcription factors were massively down-regulated in NTRK2-/- cells, while early glial progenitor markers were enriched in NTRK2-/- cells. This indicates a previously undescribed inhibitory role of TrkB on glial differentiation in addition to its well described pro-neurogenesis role. Altogether, we have generated for the first time a human neural cell line with a loss-of-function mutation of NTRK2, which represents a reproducible and readily available cell culture system to study the role of TrkB during human neural differentiation, analyse the role of TrkB isoforms as well as validate TrkB antibodies and pharmacological agents targeting the TrkB pathway. WT and NOX4 KO primary mouse skin fibroblasts were stimulated with 10ng/ml TGF-beta 2 in three biological replicates, total 12 samples were analyzed

原肌球蛋白受体激酶B（tropomyosin receptor kinase B，TrkB）由NTRK2基因编码，隶属于跨膜酪氨酸激酶家族，该家族在神经系统的发育与稳态维持中发挥关键作用。脑源性神经营养因子（Brain-derived neurotrophic factor，BDNF）以及神经营养因子NT3、NT4/5对TrkB具有高亲和力。TrkB的表达失调与广泛的疾病谱密切相关，涵盖神经退行性疾病、精神疾病及部分癌症。TrkB的功能及其在神经发育中的作用，主要通过转基因小鼠模型、药理学调节剂以及过表达NTRK2的人神经元细胞系得以解析。本研究中，我们在永生化人神经祖细胞系ReNcell VM中检测到TrkB的高表达与显著活性，并利用CRISPR/Cas9基因编辑技术构建了NTRK2敲除（NTRK2-/-）的ReNcell VM细胞系。全转录组分析结果显示，参与神经发生、神经元发育及胶质细胞分化的特定基因的表达发生了显著改变。具体而言，在NTRK2-/-细胞中，关键的神经发生相关转录因子被显著下调，而早期胶质祖细胞标志物则在该细胞系中富集。这表明，除了此前已被阐明的促神经发生功能外，TrkB还对胶质细胞分化具有此前未被报道的抑制作用。综上，我们首次构建了携带NTRK2功能丧失性突变的人神经细胞系。该细胞系可作为可重复且易于获取的细胞培养模型，用于研究TrkB在人神经分化过程中的作用、分析TrkB异构体的功能，以及验证靶向TrkB通路的抗体与药理学制剂。我们设置3次生物学重复，以10ng/ml的转化生长因子-β2（Transforming Growth Factor-β2，TGF-β2）刺激野生型（Wild Type，WT）及NOX4敲除（NOX4 KO）的原代小鼠皮肤成纤维细胞，最终共分析12份样本。