title: Silencing of PNPLA6, the neuropathy target esterase (NTE) codifying gene, alters neurodifferentiation of human embryonal carcinoma stem cells (NT2).
abstract: 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.
authors.affiliation: 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. Electronic address: dpamies@jhsph.edu.
authors.forename: D
authors.initials: D
authors.lastname: Pamies
authors.affiliation: Institute for Health and Consumer Protection, European Commission Joint Research Centre, Ispra, VA 21027, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Bal-Price
authors.affiliation: Institute for Health and Consumer Protection, European Commission Joint Research Centre, Ispra, VA 21027, Italy; Università degli Studi dell'Insubria, Department of Clinical and Experimental Medicine, Via Dunant, 5, Varese 21100, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Fabbri
authors.affiliation: Institute for Health and Consumer Protection, European Commission Joint Research Centre, Ispra, VA 21027, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Gribaldo
authors.affiliation: Institute for Health and Consumer Protection, European Commission Joint Research Centre, Ispra, VA 21027, Italy.
authors.forename: B
authors.initials: B
authors.lastname: Scelfo
authors.affiliation: 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.
authors.forename: G
authors.initials: G
authors.lastname: Harris
authors.affiliation: Institute for Health and Consumer Protection, European Commission Joint Research Centre, Ispra, VA 21027, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Collotta
authors.affiliation: Instituto de Bioingeniería, Universidad Miguel Hernández, Elche, 03202, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Vilanova
authors.affiliation: Instituto de Bioingeniería, Universidad Miguel Hernández, Elche, 03202, Spain.
authors.forename: M A
authors.initials: MA
authors.lastname: Sogorb
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000235:genetics*; D002454:Cell Differentiation / Q000235:genetics*; D054278:Embryonal Carcinoma Stem Cells; D015870:Gene Expression / Q000235:genetics*; D020868:Gene Silencing; D006801:Humans; D010740:Phospholipases / Q000235:genetics*; D000217:Acyltransferases
chemical_list: D002265:Carboxylic Ester Hydrolases; D010740:Phospholipases; C586915:PNPLA6 protein, human; D000217:Acyltransferases; C021859:neurotoxic esterase
keywords: PNPLA6; hNT2; in vitro; neurodifferentiation; neuropathy target esterase; silencing