Neurosteroid Allopregnanolone Regulates EAAC1-Mediated Glutamate Uptake and Triggers Actin Changes in Schwann Cells CARLA PEREGO, 1 * ELIANA S. DI CAIRANO, 1 MARINELLA BALLABIO, 2 AND VALERIO MAGNAGHI 2 ** 1 Department of Molecular Sciences Applied to Biosystems, Universita` degli Studi di Milano, Milan, Italy 2 Department of Endocrinology, Physiopathology and Applied Biology, Universita` degli Studi di Milano, Milan, Italy Recent evidence shows that neurotransmitters (e.g., GABA, Ach, adenosine, glutamate) are active on Schwann cells, which form myelin sheaths in the peripheral nervous system under different pathophysiologic conditions. Glutamate, the most important excitatory neurotransmitter, has been recently involved in peripheral neuropathies, thus prevention of its toxic effect is desirable to preserve the integrity of peripheral nervous system and Schwann cells physiology. Removal of glutamate from the extracellular space is accomplished by the high affinity glutamate transporters, so we address our studies to analyze their functional presence in Schwann cells. We first demonstrate that Schwann cells express the EAAC1 transporter in the plasma membrane and in intracellular vesicular compartments of the endocytic recycling pathways. Uptake experiments confirm its presence and functional activity in Schwann cells. Secondly, we demonstrate that the EAAC1 activity can be modulated by exposure to the neurosteroid allopregnanolone 10 nM (a progesterone metabolite proved to support Schwann cells). Transporter up-regulation by allopregnanolone is rapid, does not involve protein neo-synthesis and is prevented by actin depolymerization. Allopregnanolone modulation involves GABA-A receptor and PKC activation, promotes the exocytosis of the EAAC1 transporter from intracellular stores to the Schwann cell membrane, in actin-rich cell tips, and modifies the morphology of cell processes. Finally, we provide evidence that glutamate transporters control the allopregnanolone-mediated effects on cell proliferation. Our findings are the first to demonstrate the presence of a functional glutamate uptake system, which can be dynamically modulated by allopregnanolone in Schwann cells. Glutamate transporters may represent a potential therapeutic target to control Schwann cell physiology. J. Cell. Physiol. 227: 1740–1751, 2012. ß 2011 Wiley Periodicals, Inc. The identification of the signals regulating Schwann cell growth, development and maturation, under physiological or pathological conditions, is of great importance for the peripheral nervous system (PNS). Recently, a role for the neurosteroid allopregnanolone (ALLO) and/or the neurotransmitter GABA in such a control has been proposed (Magnaghi, 2007). ALLO is synthesized de novo in Schwann cells (Schumacher et al., 2000) and influences PNS myelinogenesis and myelin protein expression (Melcangi et al., 2005; Schumacher et al., 2007). At nanomolar concentrations, ALLO acts as a potent allosteric GABA-A receptor modulator (Majewska et al., 1986) and regulates respectively glutamate decarboxylase (Chavez-Delgado et al., 2005) and GABA synthesis in Schwann cells (Magnaghi et al., 2010) sustaining a GABA autocrine signaling in these cells. Among the putative factors regulating Schwann cells it should be also considered glutamate, the major excitatory neurotransmitter and potent neurotoxin in the nervous system (Choi et al., 1987). The presence of glutamate in the Schwann cells is still an intriguing but controversial issue. Some observations show that Schwann cells can release relevant concentrations of the excitotoxic glutamate (Wu et al., 2005), whereas other data reveal an immunonegativity for glutamate in the Schwann and satellite cells of the rat nodose ganglion (Schaffar et al., 1997). Schwann cells in vivo and in vitro express a variety of ionotropic (i.e., NMDA, AMPA, or KA receptors; Fink et al., 1999; Kinkelin et al., 2000) and metabotropic glutamate receptors (Saitoh and Araki, 2010) and the enzymes involved in glutamate metabolism, such as GAD (Magnaghi et al., 2010) and glutamine synthetase (GS) (Saitoh and Araki, 2010). Altogether these findings suggest a role of glutamate as a signaling molecule in the Schwann cell/neuron interactions and predict the expression of a glutamate clearance system to tightly control its concentration in the extracellular space. In the CNS, this function is accomplished by high-affinity Na- dependent glutamate transporters (excitatory amino acid transporters, EAATs) (Danbolt, 2001). These transporters include glutamate-aspartate transporter (GLAST or EAAT1), glutamate transporter 1 (GLT1 or EAAT2) and excitatory amino acid carrier 1 (EAAC1 or EAAT3). High affinity glutamate transporters have been recently identified in the PNS in vivo. GLT1 was found in the cytoplasm of Schwann cells; GLAST Additional Supporting Information may be found in the online version of this article. Contract grant sponsor: University Research Program; Contract grant number: PUR 2007/2008. Contract grant sponsor: Association Francaise contre les Myopathies (AFM); Contract grant number: 14163/2009. Contract grant sponsor: Compagnia di San Paolo (Bando Programma Neuroscienze 2008-Project MOVAG); Contract grant number: 2229/2008. *Correspondence to: Carla Perego, Department of Molecular Sciences Applied to Biosystems, Universita ` degli Studi di Milano, Via Trentacoste 2, 20134 Milan, Italy. E-mail: carla.perego@unimi.it **Correspondence to: Valerio Magnaghi, Department of Endocrinology, Physiolpathology, Applied Biology, Universita ` degli Studi di Milano, Via G. Balzaretti 9, 20133 Milan, Italy. E-mail: valerio.magnaghi@unim.it Received 1 December 2010; Accepted 10 June 2011 Published online in Wiley Online Library (wileyonlinelibrary.com), 17 June 2011. DOI: 10.1002/jcp.22898 ORIGINAL RESEARCH ARTICLE 1740 Journal of Journal of Cellular Physiology Cellular Physiology ß 2011 WILEY PERIODICALS, INC.