**1. Introduction**

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Shimomura K, Ashcroft FM, Thorens B, Rorsman P & Krek W (2008). pVHL is a regulator of glucose metabolism and insulin secretion in pancreatic beta-cells. *Genes*  Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS). In addition to functioning as a neurotransmitter at the majority of brain synapses, it is the substrate for synthesis of the major inhibitory transmitter γ-aminobutyric acid (GABA). However, glutamate is also a neurotoxin, and a number of molecular control mechanisms are responsible for maintaining extracellular glutamate below excitotoxic levels. Na+-dependent excitatory amino acid transporters (EAATs) are crucial regulators of extracellular glutamate and also act to control the dynamics of excitatory transmission in the CNS (Danbolt, 2001). The Na+-dependent excitatory amino acid carrier 1 (EAAC1) is expressed in the somata and dendrites of many neuronal types, including pyramidal cells of the hippocampal formation and cortex, and many subtypes of GABAergic inhibitory neurons (Rothstein et al., 1994). The physiological significance of EAAC1 is unclear because the subcellular distribution and kinetic properties of this transporter would not allow for a substantial contribution to glutamate clearance from the synaptic cleft; rather, these functions are mediated by glial EAATs (EAAT1 and EAAT2) located in the perisynaptic region. Recent studies have demonstrated multiple functions for EAAC1 distinct from clearance of glutamate from CNS synapses (Kiryu-Seo et al., 2006; Levenson et al., 2002; Peghini et al., 1997; Sepkuty et al., 2002). For example, decreased EAAC1 expression in the CNS impairs neuronal glutathione (GSH) synthesis, leading to oxidative stress and agedependent neurodegeneration (Aoyama et al., 2006), suggesting that aberrant EAAC1 expression contributes to the pathogenesis of neurodegenerative diseases.

Studies conducted over the past decade on the kinetics of EAAC1 and regulation of transporter expression and function have lead to a greater appreciation of the physiological and pathophysiological relevance of EAAC1 (Aoyama et al., 2008b; Danbolt, 2001; Kanai & Hediger, 2004; Nieoullon et al., 2006), but there are many issues to be resolved for a thorough understanding of the significance of EAAC1 in normal brain function and disease. In particular, the regulatory mechanisms of EAAC1-mediated glutamate uptake are largely unknown. The recent discovery of addicsin (glutamate transporter-associated protein 3-18, GTRAP3-18) as an EAAC1 binding protein has contributed greatly to our understanding of the regulatory mechanisms of EAAC1 activity (Lin et al., 2001). Furthermore, we recently proposed a regulatory model of EAAC1-mediated glutamate uptake by addicsin complexes (Akiduki & Ikemoto, 2008). In this chapter, we describe the regulation of EAAC1-mediated glutamate uptake based on our recent results. To better understand this regulatory mechanism, we first explain three key molecules involved in this regulatory pathway— EAAC1, addicsin, and ADP-ribosylation factor-like 6 interacting protein 1 (Arl6ip1).

#### **1.1 EAAC1**

The EAAC1 protein was first identified as a Na+-dependent high-affinity glutamate transporter by expression cloning in *Xenopus* oocytes (Kanai & Hediger, 1992). Stoichiometric analysis demonstrates that EAAC1 transports L-glutamate, L-aspartate, and D-aspartate, accompanied by the cotransport of 3 Na+ and 1 H+, and the countertransport of 1 K+ (Kanai & Hediger, 2003). In mammalian tissues, there are five different subtypes of EAATs—EAAT1 (glutamate/aspartate transporter, GLAST), EAAT2 (glutamate transporter 1, GLT-1), EAAT3 (EAAC1), EAAT4, and EAAT5 (Danbolt, 2001). These EAATs are structurally similar; all have eight transmembrane domains and a pore loop between the seventh and eighth domain. Most EAATs play an important role in removing extracellular glutamate from the synaptic and extrasynaptic space (Kanai & Hediger, 2003), particularly GLAST and GLT-1. These two isoforms are primarily expressed in glial cells and play a major role in protecting neurons from glutamate-induced toxicity (Rothstein et al., 1994) as well as terminating glutamatergic transmission (Rothstein et al., 1993; Tong & Jahr, 1994). In contrast, EAAC1 is diffusely localized to the cell bodies and dendrites of neurons and is enriched in cortical and hippocampal pyramidal cells as well as in some inhibitory neurons (Conti et al., 1998; Rothstein et al., 1994). This subcellular localization and restricted distribution indicate that EAAC1 does not play a major role in glutamate clearance from the synaptic cleft (Rothstein et al., 1996). Recent studies suggest that EAAC1 contributes to multiple physiological functions distinct from glutamate clearance. Indeed, EAAC1 transport provides cysteine as a substrate of GSH synthesis (Y. Chen & Swanson, 2003; Himi et al., 2003; Watabe et al., 2008; Zerangue & Kavanaugh, 1996). Neurons cannot transport extracellular GSH and therefore must transport cysteine from the extracellular space for *de novo* GSH synthesis from cysteine (Aoyama et al., 2008b). In the CNS, the depletion of GSH is associated with neurodegenerative disorders, including Alzheimer's and Parkinson's diseases (Ramassamy et al., 2000; Sian et al., 1994). Consistent with these results, EAAC1 knockout mice show oxidative stress in neurons and age-dependent neurodegeneration, pathologies that are rescued by *N*-acetylcysteine, a membrane-permeable cysteine precursor (Aoyama et al., 2006). These mice also show alteration of zinc homeostasis and increased neural damage after transient cerebral ischemia (Won et al., 2010). Furthermore, in a knockin mouse model of Huntington's disease, in which human *huntingtin* exon 1 with 140 CAG repeats was inserted into the wild-type low CGA repeat mouse *huntingtin* gene, oxidative stress and cell death were caused by abnormal Rab11-dependent EAAC1 trafficking to the cell surface (X. Li et al., 2010). In addition, 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine-treated mice, an animal model of Parkinson's disease, show reduced EAAC1-mediated neuronal cysteine uptake, impaired GSH synthesis, and motor dysfunction (Aoyama et al., 2008a). These results indicate that dysfunctional EAAC1 mediated cysteine transport increases neural vulnerability to oxidative stress and could contribute to the pathogenesis of neurodegenerative diseases.

proposed a regulatory model of EAAC1-mediated glutamate uptake by addicsin complexes (Akiduki & Ikemoto, 2008). In this chapter, we describe the regulation of EAAC1-mediated glutamate uptake based on our recent results. To better understand this regulatory mechanism, we first explain three key molecules involved in this regulatory pathway—

The EAAC1 protein was first identified as a Na+-dependent high-affinity glutamate transporter by expression cloning in *Xenopus* oocytes (Kanai & Hediger, 1992). Stoichiometric analysis demonstrates that EAAC1 transports L-glutamate, L-aspartate, and D-aspartate, accompanied by the cotransport of 3 Na+ and 1 H+, and the countertransport of 1 K+ (Kanai & Hediger, 2003). In mammalian tissues, there are five different subtypes of EAATs—EAAT1 (glutamate/aspartate transporter, GLAST), EAAT2 (glutamate transporter 1, GLT-1), EAAT3 (EAAC1), EAAT4, and EAAT5 (Danbolt, 2001). These EAATs are structurally similar; all have eight transmembrane domains and a pore loop between the seventh and eighth domain. Most EAATs play an important role in removing extracellular glutamate from the synaptic and extrasynaptic space (Kanai & Hediger, 2003), particularly GLAST and GLT-1. These two isoforms are primarily expressed in glial cells and play a major role in protecting neurons from glutamate-induced toxicity (Rothstein et al., 1994) as well as terminating glutamatergic transmission (Rothstein et al., 1993; Tong & Jahr, 1994). In contrast, EAAC1 is diffusely localized to the cell bodies and dendrites of neurons and is enriched in cortical and hippocampal pyramidal cells as well as in some inhibitory neurons (Conti et al., 1998; Rothstein et al., 1994). This subcellular localization and restricted distribution indicate that EAAC1 does not play a major role in glutamate clearance from the synaptic cleft (Rothstein et al., 1996). Recent studies suggest that EAAC1 contributes to multiple physiological functions distinct from glutamate clearance. Indeed, EAAC1 transport provides cysteine as a substrate of GSH synthesis (Y. Chen & Swanson, 2003; Himi et al., 2003; Watabe et al., 2008; Zerangue & Kavanaugh, 1996). Neurons cannot transport extracellular GSH and therefore must transport cysteine from the extracellular space for *de novo* GSH synthesis from cysteine (Aoyama et al., 2008b). In the CNS, the depletion of GSH is associated with neurodegenerative disorders, including Alzheimer's and Parkinson's diseases (Ramassamy et al., 2000; Sian et al., 1994). Consistent with these results, EAAC1 knockout mice show oxidative stress in neurons and age-dependent neurodegeneration, pathologies that are rescued by *N*-acetylcysteine, a membrane-permeable cysteine precursor (Aoyama et al., 2006). These mice also show alteration of zinc homeostasis and increased neural damage after transient cerebral ischemia (Won et al., 2010). Furthermore, in a knockin mouse model of Huntington's disease, in which human *huntingtin* exon 1 with 140 CAG repeats was inserted into the wild-type low CGA repeat mouse *huntingtin* gene, oxidative stress and cell death were caused by abnormal Rab11-dependent EAAC1 trafficking to the cell surface (X. Li et al., 2010). In addition, 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine-treated mice, an animal model of Parkinson's disease, show reduced EAAC1-mediated neuronal cysteine uptake, impaired GSH synthesis, and motor dysfunction (Aoyama et al., 2008a). These results indicate that dysfunctional EAAC1 mediated cysteine transport increases neural vulnerability to oxidative stress and could

EAAC1, addicsin, and ADP-ribosylation factor-like 6 interacting protein 1 (Arl6ip1).

contribute to the pathogenesis of neurodegenerative diseases.

**1.1 EAAC1** 

In addition to cysteine transport, EAAC1 has several other functions unrelated to removal of extracellular glutamate. For instance, EAAC1 promotes GABA synthesis by supplying the substrate glutamate (Mathews & Diamond, 2003; Sepkuty et al., 2002). Therefore, EAAC1 can strengthen inhibitory synapses in response to elevations in extracellular glutamate and contribute indirectly to GABA release (Mathews & Diamond, 2003). Indeed, a loss of EAAC1 function leads to epilepsy (Sepkuty et al., 2002), underscoring the importance of EAAC1 in GABAergic transmission. Furthermore, EAAC1 plays a crucial role in preventing neuronal death by suppressing glutamate excitotoxicity (Kiryu et al., 1995; Murphy et al., 1989) and has a mitochondria-mediated anti-apoptotic function in injured motor neurons (Kiryu-Seo et al., 2006). These studies and those discussed in Section 3.4 strongly suggest that EAAC1 contributes to multiple functions in the CNS distinct from glutamate clearance.

The regulatory mechanisms of EAAC1 have been widely investigated *in vitro*. Cumulative evidence demonstrates that glutamate uptake by EAAC1 is facilitated by cell signaling molecules and accessory proteins that promote the redistribution of EAAC1 from the endoplasmic reticulum (ER) to the plasma membrane. First, several reports demonstrate that several kinase signaling cascades regulate EAAC1 activity. In C6BU-1 glioma cells and primary neuronal cultures, phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, rapidly increases EAAC1-mediated glutamate uptake (Dowd & Robinson, 1996). This effect is regulated by mechanisms that are independent of *de novo* synthesis of new transporters but is related to the redistribution of EAAC1 from subcellular compartments to the plasma membrane (Davis et al., 1998; Fournier et al., 2004; Sims et al., 2000). Pharmacological analyses demonstrate that PKCα regulates EAAC1 translocation from intracellular compartments to the cell surface, and that PKCε increases EAAC1 functional activity (Gonzalez et al., 2002). PKCα interacts with EAAC1 in a PKC-dependent manner and phosphorylates EAAC1 (Gonzalez et al., 2003). Platelet-derived growth factor (PDGF) increases the delivery of EAAC1 to the cell surface through phosphatidylinositol 3-kinase (PI3K) activity (Fournier et al., 2004; Sheldon et al., 2006; Sims et al., 2000). Consistent with this result, wortmannin, a PI3K inhibitor, decreases cell surface expression of EAAC1 and inhibits EAAC1-mediated glutamate uptake (Davis et al., 1998). In addition, PKC and PDGF have different effects on trafficking and internalization of EAAC1; PMA, but not PDGF, reduces internalization of EAAC1 (Fournier et al., 2004). Thus, EAAC1 trafficking is regulated by two independent signaling pathways. In contrast, PKC negatively regulates EAAC1-mediated glutamate uptake in *Xenopus* oocytes (Trotti et al., 2001) and in Madin– Darby canine kidney (MDCK) cells (Padovano et al., 2009) by inhibiting cell surface expression through calcineurin-mediated internalization (Padovano et al., 2009; Trotti et al., 2001), suggesting that the regulatory mechanisms of EAAC1 surface expression and function by PKC are specific to cell type and depend on specific PKC isozymes. Second, accessory proteins regulate EAAC1 activity. For instance, δ opiod receptor interacts with EAAC1 and inhibits EAAC1-mediated glutamate uptake in *Xenopus* oocytes and rat hippocampal neurons (Xia et al., 2006). In addition, *N*-methyl-*D*-aspartate receptors containing NR1, NR2A, and/or NR2B interact with EAAC1 and facilitate the cell surface expression of EAAC1 in C6BU-1 cells and rat hippocampal neurons (Waxman et al., 2007). Moreover, the cell surface expression of EAAC1 is controlled by interactions with Na+/H+ exchanger regulatory factor 3 (NHERF-3, also called PDZK1) and adaptor protein 2 (AP-2). While NHERF-3 promotes the delivery of EAAC1 to the plasma membrane, AP-2 regulates constitutive endocytosis of EAAC1 in MDCK cells (D'Amico et al., 2010). Furthermore, reticulon 2B (RTN2B) interacts with EAAC1 and addicsin/GTRAP3-18, and promotes intracellular trafficking of EAAC1 in HEK293 cells and cultured cortical neurons (Liu et al., 2008). Addicsin/GTRAP3-18 interacts with EAAC1 and inhibits EAAC1 trafficking in HEK293 cells (Ruggiero et al., 2008). Thus, multiple regulatory mechanisms control EAAC1 trafficking and membrane expression, but the molecular details are generally unclear. In this study, we focus on the regulation of EAAC1 trafficking by addicsin.
