Pamies D, Bal-Price A, Fabbri M, Gribaldo L, Scelfo B, Harris G, Collotta A, Vilanova E, Sogorb M A
Instituto de Bioingeniería, Universidad Miguel Hernández, Elche, 03202, Spain; Center for Alternatives to Animal Testing (CAAT) at Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA; Institute for Health and Consumer Protection, European Commission Joint Research Centre, Ispra, VA 21027, Italy.
Institute for Health and Consumer Protection, European Commission Joint Research Centre, Ispra, VA 21027, Italy.
Neuroscience. 2014 Dec 5;281:54-67. doi: 10.1016/j.neuroscience.2014.08.031. Epub 2014 Sep 26.
Neuropathy target esterase (NTE) is a protein involved in the development of a polyneuropathy caused by exposure to certain organophosphorus compounds. In vivo and in vitro studies have also associated NTE with embryonic development since NTE null mice embryos are non-viable, and silencing the NTE-codifying gene (Pnpla6) in mouse embryonic stem cells strongly alters the differentiation of vascular and nervous systems. In this paper, human embryonal carcinoma stem cells human-derived NTera2/D1 (hNT2) are used as an in vitro neurodifferentiation model to determine whether PNPLA6 silencing is able to alter the differentiation process. In control cultures, PNPLA6 mRNA levels increased in parallel with other neuroectodermal markers during neurodifferentiation. PNPLA6 silencing with specific interference RNA reached a 97% decrease in gene expression 3days after transfection and with a maximum reduction in NTE enzymatic activity (50%), observed on day 4. Silencing PNPLA6 showed an 80% decrease in quantifiable neuronal cells after 13days in vitro (DIV) compared to controls and absence of different neuronal markers after 66DIV. Microarray data analysis of the PNPLA6-silenced cells showed alterations in several developmental processes, mainly neurogenesis and epithelium tube morphogenesis. PNPLA6 silencing also led to a reduction in electrical activity and an altered neuronal phenotype. This work is the first proof supporting the hypothesis that NTE plays a role in human early neurodevelopment using a human cell differentiation model.
神经病靶酯酶（NTE）是一种参与由接触某些有机磷化合物引起的多发性神经病发展过程的蛋白质。体内和体外研究也将NTE与胚胎发育联系起来，因为NTE基因敲除小鼠胚胎无法存活，并且在小鼠胚胎干细胞中沉默编码NTE的基因（Pnpla6）会强烈改变血管和神经系统的分化。在本文中，人胚胎癌细胞系人源NTera2/D1（hNT2）被用作体外神经分化模型，以确定Pnpla6沉默是否能够改变分化过程。在对照培养物中，在神经分化过程中，Pnpla6 mRNA水平与其他神经外胚层标志物平行增加。用特异性干扰RNA沉默Pnpla6后，转染3天后基因表达下降了97%，在第4天观察到NTE酶活性最大降低（50%）。与对照相比，沉默Pnpla6后，体外培养13天（DIV）时可量化的神经元细胞减少了80%，66DIV后不同神经元标志物缺失。对沉默Pnpla6的细胞进行微阵列数据分析显示，几个发育过程发生了改变，主要是神经发生和上皮管形态发生。Pnpla6沉默还导致电活动减少和神经元表型改变。这项工作首次证明了使用人类细胞分化模型支持NTE在人类早期神经发育中起作用这一假设。
