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SVCT2 Overexpression in Neuroblastoma Cells Induces Cellular Branching that is Associated with ERK Signaling

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Abstract

Expression of the sodium and ascorbic acid (AA) cotransporter SVCT2 is induced during the period of cellular arborization and synaptic maturation of early postnatal (P1-P5) rat cerebral neurons. The physiological importance of the transporter for neurons is evidenced by the lethality and delayed neuronal differentiation detected in mice with ablation of SVCT2. The mechanism(s) involved in these defects and the role of SVCT2 in neuronal branching have not been determined yet. To address this, we used lentiviral expression vectors to increase the levels of SVCT2 in N2a cells and analyzed the effects on neurite formation. Expression of a fusion protein containing the human SVCT2wt and EYFP induced an increase in the number of MAP2+ neurites and filopodia in N2a cells. Overexpression of SVCT2 and treatment with AA promoted ERK1/2 phosphorylation. Our data suggest that enhanced expression of the high affinity AA transporter SVCT2, which tightly regulates intracellular AA concentrations, induces neuronal branching that then activates key signaling pathways that are involved in the differentiation and maturation of cortical neurons during postnatal development.

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Abbreviations

AA:

Ascorbic acid

DMEM:

Dulbecco’s modified Eagle’s medium

EYFP:

Enhanced yellow fluorescent protein

FBS:

Fetal bovine serum

GLUTs:

Glucose transporters

PAGE:

Polyacrylamide gel electrophoresis

PBS:

Phosphate-buffered saline

SDS:

Sodium dodecyl sulfate

SVCT:

Sodium and ascorbic acid cotransporter

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Acknowledgments

This work was supported by a FONDECYT grant, 1140477, and a CONICYT PIA ECM-12 grant (both to Francisco Nualart) and an NIH grant (RNS065808A) to Ernesto R. Bongarzone. The funders had no role in study design, data collection and analysis, the decision to publish, or preparation of the manuscript.

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Authors and Affiliations

  1. Laboratory of Neurobiology and Stem Cells, Center for Advanced Microscopy CMA BIO BIO, Faculty of Biological Sciences, University of Concepcion, Concepción, Chile

    Katterine Salazar, Milka Martínez, Viviana Ulloa, Romina Bertinat, Fernando Martínez, Nery Jara, Francisca Espinoza & Francisco Nualart

  2. Department of Anatomy and Cell Biology, College of Medicine, University of Illinois Chicago, Chicago, IL, USA

    Ernesto R. Bongarzone

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  1. Katterine Salazar
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Correspondence to Francisco Nualart.

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Supplementary Figure 1

SVCT2 lentiviral overexpression in N2a cells. Vectors used to produce the lentiviral particles in the HEK293T cell line (A). Western blot analysis of EGFP expression in total protein extracts obtained from non-transduced (lane 1), EGFP-transduced (lane 2) and hSVCT2-EYFP-transduced (lane 3) cells (B). Confocal microscopy analysis in cells transduced with hSVCT2-EYFP lentivirus, showing the outgrowth of processes (arrows) and filopodia (arrowhead). Nuclei were stained with TOPRO-3 (blue channel) (D-F). (DOCX 1484 kb)

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Salazar, K., Martínez, M., Ulloa, V. et al. SVCT2 Overexpression in Neuroblastoma Cells Induces Cellular Branching that is Associated with ERK Signaling. Mol Neurobiol 53, 6668–6679 (2016). https://doi.org/10.1007/s12035-015-9553-z

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