Glycogen synthase kinase GSK is currently considered to be a

Glycogen synthase kinase 3 (GSK3) is currently considered to be a multifunctional serine/threonine kinase involved in a wide spectrum of cellular processes such as glycogen metabolism, cell proliferation, neuronal function, oncogenesis or embryonic development (for recent reviews see: Rayasam et al., 2009, Wildburger and Laezza, 2012). Although the protein is expressed in nearly all tissues, its highest levels and activity are found in the CNS (Leroy and Brion, 1999, Woodgett, 1990). Two distinct, but closely related forms of GSK3, GSK3α and GSK3β, have been identified. GSK3 is constitutively active in resting Sabutoclax and its activity can be inhibited by phosphorylation at serine residues (Ser21 for GSK3α and Ser9 for GSK3β) on their N-terminal domain. By controlling the phosphorylation of these residues, neurons regulate GSK3 activity (for review see Doble and Woodgett, 2003). In animal models, the overexpression of GSK-3 induces increased vulnerability to mood-related behavioral disturbances and impaired socialization behavior (Mines et al., 2010, Polter et al., 2010). Furthermore, in clinical studies changes in the expression and activity of GSK-3 are found in schizophrenia (Emamian, 2012, Jope, 2003, Kozlovsky et al., 2001, Kozlovsky et al., 2002, Lovestone et al., 2007), mood disorders (Eldar-Finkelman, 2002, Jope, 2011), addictive behaviors (Miller et al., 2009, Miller et al., 2010) and Alzheimer's disease (Balaraman et al., 2006, Hooper et al., 2008, Kremer et al., 2011). Recently, the role of GSK3β has emerged in the pathogenesis of pain (Maixner et al., 2014). Despite the pleiotropic effects of GSK3, or probably because of them, many of their molecular targets in the CNS have not yet been identified. Lately, GSK-3 has been proposed as a key element in plasticity at excitatory and inhibitory synapses in the CNS (Bradley et al., 2012). The molecular mechanisms underlying, at least partially, the role of GSK-3 in synaptic plasticity is through the regulation of NMDA and AMPA receptors endocytosis (Bradley et al., 2012, Chen et al., 2007, Wei et al., 2010).
Materials and methods
Results
Discussion The present study reveals a completely novel function of GSK3β, the differential modulation of the Na+-coupled glycine transporters GlyT1 and GlyT2. Considering that the function of these proteins is critical for the proper functioning of glycinergic and the NMDA-mediated glutamatergic synapses, the results of this work increase the knowledge of the regulatory mechanisms of excitatory and inhibitory neurotransmission in the CNS with a potential pathophysiological impact. In this work we have shown that the co-expression of GSK3β with GlyT1 or GlyT2 leads to a down- and up-regulation of the transporter activity, respectively, in two different heterologous expression systems, COS-7 cells and X. laevis oocytes. These functional changes are consistent with a decrease and increase of the GlyT1 and GlyT2 levels at the plasma membrane, respectively. The specificity of these changes is supported by the antagonism exerted by a catalytically inactive form of the kinase and through inhibitors of GSK3β kinase, such as TDZD-8 and lithium. Additional evidence of the opposite modulation carried out by the overexpression of GSK3β on GlyT1 and GlyT2, are provided by the results of the pharmacological inhibition of GSK3β in neuron primary cultures of brainstem and spinal cord. Both, the activity and cell surface expression of the endogenous transporters showed changes as a result of neuronal kinase inhibition. Altogether, the different modulation of GlyT1 and GlyT2 exerted by GSK3β in neuronal cultures and in cells expressing the recombinant proteins indicates that the observed glycine transport regulation is indeed the consequence of specific effects on the transporters. Glycogen synthase kinase 3 (GSK3β) is highly expressed in the CNS and is considered to be a multifunctional serine/threonine kinase involved in neuronal development, mood stabilization, and neurodegeneration. Recently it has been proposed that GSK3β plays a major role in plasticity at excitatory and inhibitory synapses in the CNS (Bradley et al., 2012). The underlying molecular mechanisms involved are, at least partially, via the regulation of the function and trafficking of NMDA and AMPA receptors (Chen et al., 2007, Bradley et al., 2012, Wei et al., 2010). Data from this work add an additional control point of glutamatergic neurotransmission by GSK3β. Considering that GlyT1 is the main regulator of glycine availability near to NMDA receptors, the modulation of its activity and cell surface presence by GSK3β could indirectly modulate the NMDAR-mediated glutamatergic synapses. Moreover, GlyT2 is the main supplier of glycine for constitutive vesicle refilling through active reuptake of the neurotransmitter to the terminal. This process is absolutely crucial to preserve the quantal glycine content inside the synaptic vesicles and is critical for regulating inhibitory synaptic strength (Gomeza et al., 2003b, Rousseau et al., 2008, Apostolides and Trussell, 2013). Therefore, because GlyT2 activity regulates the glycinergic synaptic strength, the new modulatory mechanism by GSK3β described here is of great physiological relevance. The results of this work suggest that constitutively active endogenous GSK3β is important for stabilizing and/or controlling the expression of functional GlyTs on the neural cell surface. The data described here and elsewhere (Chen et al., 2007, Bradley et al., 2012, Wei et al., 2010) could represent a more general process underlying CNS synaptic plasticity whereby GSK3β controls neuronal proteins that are crucial for the proper functioning of inhibitory and excitatory fast neurotransmission.