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Functional pathways altered after silencing Pnpla6 (the codifying gene of neuropathy target esterase) in mouse embryonic stem cells under differentiation

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Abstract

Neuropathy target esterase (NTE) is involved in several disorders in adult organisms and embryos. A relationship between NTE and nervous system integrity and maintenance in adult systems has been suggested. NTE-related motor neuron disease is associated with the expression of a mutant form of NTE and the inhibition and further modification of NTE by organophosphorus compounds is the trigger of a delayed neurodegenerative neuropathy. Homozygotic NTE knockout mice embryos are not viable, while heterozygotic NTE knockout mice embryos yields mice with neurological disorders, which suggest that this protein plays a critical role in embryonic development. The present study used D3 mouse embryonic stem cells with the aim of gaining mechanistic insights on the role of Pnpla6 (NTE gene encoding) in the developmental process. D3 cells were silenced by lipofectamine transfection with a specific interference RNA for Pnpla6. Silencing Pnpla6 in D3 monolayer cultures reduced NTE enzymatic activity to 50% 20 h post-treatment, while the maximum loss of Pnpla6 expression reached 80% 48 h postsilencing. Pnpla6 was silenced in embryoid bodies and 545 genes were differentially expressed regarding the control 96 h after silencing, which revealed alterations in multiple genetic pathways, such as cell motion and cell migration, vesicle regulation, and cell adhesion. These findings also allow considering that these altered pathways would impair the formation of respiratory, neural, and vascular tubes causing the deficiencies observed in the in vivo development of nervous and vascular systems. Our findings, therefore, support the previous observations made in vivo concerning lack of viability of mice embryos not expressing NTE and help to understand the biology of several neurological and developmental disorders in which NTE is involved.

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Notes

  1. Throughout the manuscript, the term NTE is always used for calling the protein called neuropathy target esterase; the term NTE enzymatic activity is used for calling the phenyl valerate esterase activity associated to NTE; while the term Pnpla6 is used for calling the NTE-codifying gene.

References

  • Akassoglou K.; Malester B.; Xu J. L.; Tessarollo L.; Rosenbluth J.; Chao M. V. Brain-specific deletion of neuropathy target esterase/Swiss cheese results in neurodegeneration. Proc. Natl. Acad. Sci. U. S. A. 101: 5075–5080; 2004.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Bautch V. L.; James J. M. Neurovascular development: the beginning of a beautiful friendship. Cell Adhes. Migr. 3: 199–204; 2009.

    Article  Google Scholar 

  • Birgbauer E.; Chun J. New developments in the biological functions of lysophospholipids. Cell. Mol. Life Sci. 63: 2695–2701; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Burnstock G. Purinergic signaling and vascular cell proliferation and death. Arterioscler. Thromb. Vasc. Biol. 22: 364–373; 2002.

    Article  PubMed  CAS  Google Scholar 

  • Camon E.; Barrell D.; Lee V.; Dimmer E.; Apweiler R. The Gene Ontology Annotation (GOA) database—an integrated resource of GO annotations to the UniProt Knowledgebase. In Silico Biol. 4: 5–6; 2004.

    PubMed  Google Scholar 

  • Chang P. A.; Chen R.; Wu Y. J. Reduction of neuropathy target esterase does not affect neuronal differentiation, but moderate expression induces neuronal differentiation in human neuroblastoma (SK-N-SH) cell line. Brain Res. Mol. Brain Res. 141: 30–38; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Chang P. A.; Sun Q.; Ni X. M.; Qv F. Q.; Wu Y. J.; Song F. Z. Molecular cloning and expression analysis of cDNA ends of chicken neuropathy target esterase. Chem. Biol. Interact. 172: 54–62; 2008.

    Article  PubMed  CAS  Google Scholar 

  • Chomczynski P.; Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156–159; 1987.

    Article  PubMed  CAS  Google Scholar 

  • Dechant G. A.; Rodríguez-Tébar A.; Barde Y. A. Neurotrophin receptors. Prog. Neurobiol. 42: 249–254; 1994.

    Article  Google Scholar 

  • Ferrri G.; Cook B. D.; Terushkin V.; Pintucci G.; Mignatti P. Transforming growth factor-beta 1 (TGF-beta 1) induces angiogenesis through vascular endothelial growth factor (VEGF)-mediated apoptosis. J. Cell. Physiol. 219: 449–458; 2009.

    Article  CAS  Google Scholar 

  • Glynn P. NTE: one target protein for different toxic syndromes with distinct mechanisms. Bioessays 25: 742–745; 2003.

    Article  PubMed  Google Scholar 

  • Greiner A. J.; Richardson R. J.; Worden R. M.; Ofoli R. Y. Influence of lysophospholipid hydrolysis by the catalytic domain of neuropathy target esterase on the fluidity of bilayer lipid membranes. Biochim. Biophys. Acta 1798: 1533–1539; 2010.

    Article  PubMed  CAS  Google Scholar 

  • Hein N. D.; Stuckey J. A.; Rainier S. R.; Fink J. K.; Richardson R. J. Constructs of human neuropathy target esterase catalytic domain containing mutations related to motor neuron disease have altered enzymatic properties. Toxicol. Lett. 196: 67–73; 2010.

    Article  PubMed  CAS  Google Scholar 

  • Hong M. S.; Hong S. J.; Barhoumi R.; Burghardt R. C.; Donnelly K. C.; Wild J. R.; Venkatraj V.; Tiffany-Castiglioni E. Neurotoxicity induced in differentiated SK-N-SH-SY5Y human neuroblastoma cells by organophosphorus compounds. Toxicol. Appl. Pharmacol. 186: 110–118; 2003.

    Article  PubMed  CAS  Google Scholar 

  • Howard A. S.; Bucelli R.; Jett D. A.; Bruun D.; Yang D.; Lein P. J. Chlorpyrifos exerts opposing effects on axonal and dendritic growth in primary neuronal cultures. Toxicol. Appl. Pharmacol. 207: 112–124; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Ip N. Y.; Yancopoulos G. D. Neurotrophic factors and their receptors. Ann. Neurol. 35(Suppl): S13–S16; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Johnson M. K. The delayed neurotoxic effect of some organophosphorus compounds: identification of the phosphorylation site as an esterase. Biochem. J. 114: 711–717; 1969.

    PubMed Central  PubMed  CAS  Google Scholar 

  • Jones F. S.; Jones P. L. The tenascin family of ECM glycoproteins: structure, function, and regulation during embryonic development and tissue remodeling. Dev. Dyn. 218: 235–259; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Kretzschmar D.; Hasan G.; Sharma S.; Heisenberg M.; Benzer S. The swiss cheese mutant causes glial hyperwrapping and brain degeneration in Drosophila. J. Neurosci. 17: 7425–7432; 1997.

    PubMed  CAS  Google Scholar 

  • Li W.; Casida J. E. Organophosphorus neuropathy target esterase inhibitors selectively block outgrowth of neurite-like and cell processes in cultured cells. Toxicol. Lett. 98: 139–146; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Li Z.; Szurek P. F.; Jiang C.; Pao A.; Bundy B.; Le W. D.; Bradley A.; Yu Y. E. Neuronal differentiation of NTE-deficient embryonic stem cells. Biochem. Biophys. Res. Commun. 30: 1103–1109; 2005.

    Article  CAS  Google Scholar 

  • Lim Y.; Matsui W. Hedgehog signaling in hematopoiesis. Crit. Rev. Eukaryot. Gene Expr. 20: 129–139; 2010.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Livak K. J.; Schmittgen T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 25: 402–408; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Lubarsky B.; Krasnow M. A. Tube morphogenesis: making and shaping biological tubes. Cell 112: 19–28; 2003.

    Article  PubMed  CAS  Google Scholar 

  • Lush M. J.; Li Y.; Read D. J.; Willis A. C.; Glynn P. Neuropathy target esterase and a homologous Drosophila neurodegeneration-associated mutant protein contain a novel domain conserved from bacteria to man. Biochem. J. 332: 1–4; 1998.

    PubMed Central  PubMed  CAS  Google Scholar 

  • Massicotte C.; Jortner B. S.; Ehrich M. Morphological effects of neuropathy-inducing organophosphorus compounds in primary dorsal root ganglia cell cultures. Neurotoxicology 24: 787–796; 2003.

    Article  PubMed  CAS  Google Scholar 

  • Moser M.; Li Y.; Vaupel K.; Kretzschmar D.; Kluge R.; Glynn P.; Buettner R. Placental failure and impaired vasculogenesis result in embryonic lethality for neuropathy target esterase-deficient mice. Mol. Cell. Biol. 24: 1667–1679; 2004.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Mühlig-Versen M.; da Cruz A. B.; Tschäpe J. A.; Moser M.; Büttner R.; Athenstaedt K.; Glynn P.; Kretzschmar D. Loss of Swiss cheese/neuropathy target esterase activity causes disruption of phosphatidylcholine homeostasis and neuronal and glial death in adult Drosophila. J. Neurosci. 25: 2865–2873; 2005.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Nakao T.; Ishizawa A.; Ogawa R. Observations of vascularization in the spinal cord of mouse embryos, with special reference to development of boundary membranes and perivascular spaces. Anat. Rec. 221: 663–677; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Pamies D.; Reig J. A.; Vilanova E.; Sogorb M. A. Expression of neuropathy target esterase in mouse embryonic stem cells during differentiation. Arch. Toxicol. 84: 481–491; 2010.

    Article  PubMed  CAS  Google Scholar 

  • Quistad G. B.; Barlow C.; Winrow C. J.; Sparks S. E.; Casida J. E. Evidence that mouse brain neuropathy target esterase is a lysophospholipase. Proc. Natl. Acad. Sci. U. S. A. 100: 7983–7987; 2003.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Rainier S.; Albers J. W.; Dyck P. J.; Eldevik O. P.; Wilcock S.; Richardson R. J.; Fink J. K. Motor neuron disease due to neuropathy target esterase gene mutation: clinical features of the index families. Muscle Nerve 43: 19–25; 2011.

    Article  PubMed  Google Scholar 

  • Read D. J.; Li Y.; Chao M. V.; Cavanagh J. B.; Glynn P. Neuropathy target esterase is required for adult vertebrate axon maintenance. J. Neurosci. 29: 11594–11600; 2009.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Read D. J.; Li Y.; Chao M. V.; Cavanagh J. B.; Glynn P. Organophosphates induce distal axonal damage, but not brain oedema, by inactivating neuropathy target esterase. Toxicol. Appl. Pharmacol. 245: 108–115; 2010.

    Article  PubMed  CAS  Google Scholar 

  • Ross B. M.; Kish S. J. Characterization of lysophospholipid metabolizing enzymes in human brain. J. Neurochem. 63: 1839–1848; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Sachana M.; Flaskos J.; Alexaki E.; Hargreaves A. J. Inhibition of neurite outgrowth in N2a cells by leptophos and carbaryl: effects on neurofilament heavy chain, GAP-43 and HSP-70. Toxicol. In Vitro 17: 115–120; 2003.

    Article  PubMed  CAS  Google Scholar 

  • Sanford L. P.; Ormsby I.; Gittenberger-de Groot A. C.; Sariola H.; Friedman R.; Boivin G. P.; Cardell E. L.; Doetschman T. TGFbeta2 knockout mice have multiple developmental defects that are non-overlapping with other TGFbeta knockout phenotypes. Development 124: 2659–2670; 1997.

    PubMed Central  PubMed  CAS  Google Scholar 

  • Schmidt A.; Wolde M.; Thiele C.; Fest W.; Kratzin H.; Podtelejnikov A. V.; Witke W.; Huttner W. B.; Söling H. D. Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid. Nature 401: 133–141; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Simon M. C.; Keith B. The role of oxygen availability in embryonic development and stem cell function. Nat. Rev. Mol. Cell Biol. 9: 285–296; 2008.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Slotkin T.; Seidler F. Transcriptional profiles reveal similarities and differences in the effects of developmental neurotoxicants on differentiation into neurotransmitter phenotypes in PC12 cells. Brain Res. Bull. 78: 211–225; 2009.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Smyth G.K.; Ritchie M.; Thorne N.; Wettenhall J. Limma: Linear models for microarray data user’s guide. Available: http://www.lcg.unam.mx/~lcollado/R/resources/limma-usersguide.pdf. 2007. Accessed February 2013.

  • Song F.; Zou C.; Han X.; Zeng T.; Zhang C.; Xie K. Reduction of retrograde axonal transport associated-proteins motor proteins, dynein and dynactin in the spinal cord and cerebral cortex of hens by tri-ortho-cresyl phosphate (TOCP). Neurochem. Int. 60: 99–104; 2012.

    Article  PubMed  CAS  Google Scholar 

  • Song Y.; Wang M.; Mao F.; Shao M.; Zhao B.; Song Z.; Shao C.; Gong Y. Knockdown of Pnpla6 protein results in motor neuron defects in zebrafish. Dis. Model Mech. 6: 404–413; 2013.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Thorsteinsdóttir S.; Deries M.; Cachaço A. S.; Bajanca F. The extracellular matrix dimension of skeletal muscle development. Dev. Biol. 354: 191–207; 2011.

    Article  PubMed  CAS  Google Scholar 

  • Vose S. C.; Fujioka K.; Gulevich A. G.; Lin A. Y.; Holland N. T.; Casida J. E. Cellular function of neuropathy target esterase in lysophosphatidylcholine action. Toxicol. Appl. Pharmacol. 232: 376–383; 2008.

    Article  PubMed  CAS  Google Scholar 

  • Wang A.; Dennis E. A. Mammalian lysophospholipases. Biochim. Biophys. Acta 1439: 1–16; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Williams D. G. Intramolecular group transfer is a characteristic of neurotoxic esterase and is independent of the tissue source of the enzyme—a comparison of the aging behavior of diisopropyl phosphorofluoridate-labeled proteins in brain, spinal-cord, liver, kidney and spleen from hen and in human-placenta. Biochem. J. 209: 817–829; 1983.

    PubMed Central  PubMed  CAS  Google Scholar 

  • Wilson P. A.; Gardner S. D.; Lambie N. M.; Commans S. A.; Crowther D. J. Characterization of the human patatin-like phospholipase family. J. Lipid Res. 47: 1940–1949; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Winrow C. J.; Hemming M. L.; Allen D. M.; Quistad G. B.; Casida J. E.; Barlow C. Loss of neuropathy target esterase in mice links organophosphate exposure to hyperactivity. Nat. Genet. 33: 477–485; 2003.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors would like to express gratitude to Drs. Luis Alcaraz and Maria Isabel Carreres (Bioarrays SL; www.bioarray.es) for their help and support in the analysis the data obtained from the microarrays.

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Authors and Affiliations

  1. Unidad de Toxicología y Seguridad Química, Instituto de Bioingeniería, Universidad Miguel Hernández de Elche, Avenida de la Universidad s/n, 03202, Elche, Spain

    David Pamies, Eugenio Vilanova & Miguel A. Sogorb

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  1. David Pamies
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  2. Eugenio Vilanova
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  3. Miguel A. Sogorb
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Correspondence to Miguel A. Sogorb.

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Editor: T. Okamoto

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ESM 1

PDF 35.5 kb

ESM 2

Gene Set Analysis Heat Map. Heat map represents the overexpressed genes (red) and the underexpressed genes (green) in the D3 EBs cultured after Pnpla6 silencing (JPEG 33 kb)

High-Resolution Image (TIFF 62.5 kb)

ESM 3

List of the 545 genes with an altered expression at 96 h after Pnpla6 silencing in the D3 embryoid bodies (PDF 77.6 kb)

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(PDF 23.6 kb)

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ESM 7

Relationships among genes involved in nervous system development and vasculogenesis. The picture shows the relationships among the 96 genes with altered expression 96 h after transfection of D3 embryoid bodies with iRNA for silencing Pnpla6. The picture was obtained using Ingenuity Pathways Analysis database (PDF 14.7 kb)

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Pamies, D., Vilanova, E. & Sogorb, M.A. Functional pathways altered after silencing Pnpla6 (the codifying gene of neuropathy target esterase) in mouse embryonic stem cells under differentiation. In Vitro Cell.Dev.Biol.-Animal 50, 261–273 (2014). https://doi.org/10.1007/s11626-013-9691-4

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