Abstract
Dendritic development is essential for the establishment of a functional nervous system. Among factors that control dendritic development, brain-derived neurotrophic factor (BDNF) has been shown to regulate dendritic length and complexity of cortical neurons. However, the cellular and molecular mechanisms that underlie these effects remain poorly understood. In this study, we examined the role of amino acid transport in mediating the effects of BDNF on dendritic development. We show that BDNF increases System A amino acid transport in cortical neurons by selective up-regulation of the sodium-coupled neutral amino acid transporter (SNAT)1. Up-regulation of SNAT1 expression and System A activity is required for the effects of BDNF on dendritic growth and branching of cortical neurons. Further analysis revealed that induction of SNAT1 expression and System A activity by BDNF is necessary in particular to enhance synthesis of tissue-type plasminogen activator, a protein that we demonstrate to be essential for the effects of BDNF on cortical dendritic morphology. Together, these data reveal that stimulation of neuronal differentiation by BDNF requires the up-regulation of SNAT1 expression and System A amino acid transport to meet the increased metabolic demand associated with the enhancement of dendritic growth and branching.
Highlights
Development of the nervous system proceeds through a sequence of complex ontogenetic processes that includes cell proliferation, migration, neurite outgrowth, axon guidance, and synapse formation [1]
Further analysis revealed that induction of SNAT1 expression and System A activity by brain-derived neurotrophic factor (BDNF) is necessary in particular to enhance synthesis of tissue-type plasminogen activator, a protein that we demonstrate to be essential for the effects of BDNF on cortical dendritic morphology
We found that treatment of cortical neurons with BDNF caused a marked increase in MeAIB uptake (Fig. 1)
Summary
Cell Culture—All experiments were performed in accordance with the European Communities Council Directive regarding the care and use of animals for experimental procedures. Cells were plated at a density of 55,000/cm on culture plates or glass coverslips in Neurobasal medium supplemented with B27 (Invitrogen, Basel, Switzerland) and 500 M glutamine. When the effects of SNAT1 siRNA and control (Ctrl) siRNA were tested, cortical neurons were cotransfected 2 days after plating with the enhanced green fluorescent protein (EGFP) expression vector pEGFP (0.1 g/well; Clontech, Basel, Switzerland) together with SNAT1 siRNA or Ctrl siRNA (0.5 g/well). [14C]␣-(Methylamino)isobutyric Acid ([14C]MeAIB) and [3H]Glutamine Uptake—After 3– 6 days in vitro, cortical neurons grown on 35 ϫ 10-mm plates were treated with 10 ng/ml BDNF in HEPES buffer (in mM: 10 HEPES, pH 7.0; 140 NaCl; 7.5 NaHCO3; 2 KCl; 2 CaCl2; 3 MgSO4, 2 KH2PO4; 5 glucose). Statistical Analysis—Data were analyzed for statistical significance by using one-way analysis of variance followed by Bonferroni post hoc test except for time course analysis of MeAIB uptake where one-way analysis of variance was followed by Dunnett post hoc test
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