Modulation of EAAC1-Mediated Glutamate Uptake by Addicsin

activity.

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 PKC regulates EAAC1 translocation from intracellular compartments to the cell surface, and that PKC increases EAAC1 functional activity (Gonzalez et al., 2002). PKC 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, 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 . Addicsin/GTRAP3-18 interacts with EAAC1 and inhibits EAAC1 trafficking in HEK293 cells . 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.

Addicsin
In many papers, human addicsin and rat addicsin are called JWA and GTRAP3-18, respectively. Addicsin, GTRAP3-18, and JWA have been independently identified by several research groups (Ikemoto et al., 2002;Lin et al., 2001;Zhou et al., GeneBank, AF070523, unpublished observations). We first identified addicsin as a novel mRNA encoding a 22-kDa hydrophobic protein that is highly expressed in the basomedial nucleus of the mouse amygdala following repeated morphine administration (Ikemoto et al., 2002). Meanwhile, GTRAP3-18 cDNA was identified as encoding an EAAC1 binding protein by yeast twohybrid screening of a rat brain cDNA library using the C-terminal intracellular domain of EAAC1 as bait (Lin et al., 2001). The JWA gene was identified as an all-trans retinoic acid (RA)-responsive factor from human tracheobronchial epithelial cells (Zhou et al.,GeneBank,AF070523,unpublished observations). Bioinformatic analysis demonstrates that JWA has a prenylated Rab acceptor 1 (PRA1) domain and 62% similarity with Jena-Muenchen 4 (JM4), a protein recently identified as PRA1 domain family member 2 (PRAF2) (Schweneker et al., 2005). Proteins containing a large PRA1 domain form a new family of PRA1 domain family proteins (PRAFs) that regulate intracellular protein trafficking. Thus, addicsin is a new member of the PRAF family, PRAF3.
The addicsin cDNA is approximately 1.4 kbp and consists of a 564-bp single open reading frame (Ikemoto et al., 2002). The addicsin gene contains three exons separated by two introns, and the sequence is highly conserved among vertebrates (Butchbach et al., 2002). Furthermore, addicsin is located on mouse chromosome 6, a location corresponding to human chromosome 3p (Butchbach et al., 2002;Ikemoto et al., 2002).
Expression profiles of addicsin and addicsin mRNA were investigated in the developing and mature brain. In the developing rat brain, the expression levels of addicsin decrease significantly from embryonic day 17 to post-natal day 0 (Maier et al., 2009). Meanwhile, addicsin mRNA levels increase gradually during early maturation, peaking around postnatal day 5, and then declining by about 50% by post-natal day 14 (Inoue et al., 2005). This developmental expression pattern corresponds to periods of elevated synaptogenesis, suggesting that addicsin is involved in synapse formation. Indeed, later in this chapter, we discuss evidence that addicsin participates in intracellular protein trafficking of neurotransmitter receptors. Addicsin is widely distributed in the brain (Akiduki et al., 2007;Butchbach et al., 2002). In the mature CNS, addicsin is expressed in the cerebral cortex, amygdala, striatum, hippocampus (CA1-3 fields), dentate gyrus, and cerebellum. Addicsin is expressed in the somata of glutamatergic and GABAergic neurons and exhibits presynaptic localization in restricted regions such as CA3 stratum lucidum (Akiduki et al., 2007). In situ hybridization analysis reveals that addicsin mRNA is widely distributed in the brain, predominantly expressed in principal neurons, including glutamatergic and GABAergic neurons in the mature CNS (Inoue et al., 2005). However, the precise subcellular localization of addicsin remains controversial. Recent reports found that addicsin is an integral ER membrane protein that prevents EAAC1 maturation and function by inhibiting ER trafficking . However, our protein fractionation analysis using mouse whole brain lysates prepared in PBS, NaCl, or Na 2 CO 3 buffer, all indicate that addicsin is predominantly present in the S1 soluble fraction, while the ER transmembrane protein calnexin is present in the P2 pellet fraction (Ikemoto et al., 2002). Our subcellular fractionation analysis with highly purified synaptic fractions prepared from mouse forebrain also support the notion that addicsin is present in the cytoplasmic and presynaptic membrane fractions (Akiduki et al., 2007). Furthermore, immunocytochemical studies reveal that addicsin is present in both the plasma membrane and the intracellular compartments, including the ER (Ikemoto et al., 2002;Watabe et al., 2007Watabe et al., , 2008. Consistent with these findings, bioinformatic analysis demonstrates that the -helix is not long enough for a transmembrane domain; nevertheless, addicsin is predicted to be a hydrophobic protein composed of 62% -helix and 8% -sheet (Butchbach et al., 2002), suggesting that it is membrane-associated. Further investigations are needed to clarify the subcellular localization of addicsin, but it is apparent that this protein can exist in both soluble and membrane-associated forms.
These potential functions remain largely speculative, but molecular studies have provided several intriguing candidates (Fig. 2). First, addicsin is involved in apoptosis induced by  retinamide, arsenic trioxide, and cadmium Zhou et al., 2008). Knockdown of addicsin attenuates all-trans RA-induced and arsenic trioxide-induced apoptosis Zhou et al., 2008). Therefore, addicsin serves as a pro-apoptotic molecule. Second, addicsin acts as an environmental stress sensor to protect cells from oxidative stress and subsequent genomic damage. Addicsin is also involved in cellular responses to environmental stresses, including oxidative stress and heat shock, and in the differentiation of leukemia cells under nonphysiological conditions Huang et al., 2006aHuang et al., , 2006bT. Zhu et al., 2005). Addicsin is upregulated after exposure to the pro-oxidants benzo[ ]pyrene and hydrogen peroxide through activation of the nuclear transcription factor I (NFI) (R. . Addicsin facilitates DNA repair by interacting with X-ray cross-complementing group 1 protein, a regulator of the DNA base excision repair processes that translocates to the nucleus in response to oxidative stress (R. Wang et al., 2009). Thus, NFImediated addicsin upregulation protects against DNA damage induced by benzo[ ]pyrene and hydrogen peroxide. Third, addicsin also inhibits cancer cell migration as was observed in HeLa, B16, and HCCLM3 cancer cells. (H. . Addicsin has an important role in maintaining the stability of F-actin and in the initiation of actin cytoskeletal rearrangements. Moreover, knockdown of addicsin results in the inactivation of the MEK-ERK signaling cascade. Thus, addicsin inhibits cell migration by activating the mitogenactivated protein kinase (MAPK) cascade and regulating the rearrangement of the F-actin cytoskeleton (H. . Fourth, addicsin participates in the regulation of GSH synthesis; the association of addicsin with EAAC1 at the plasma membrane inhibits the uptake of cysteine for GSH synthesis and thus determines the intracellular GSH content in vitro and in vivo (Watabe et al., 2007. This suggests that addicsin is a therapeutic target for enhancing GSH levels in patients with neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases, associated with oxidative stress. Fifth, addicsin significantly inhibits neurite growth in differentiated CAD cells by inactivating Rab1, a positive regulator of ER-to-Golgi trafficking (Maier et al., 2009). Finally, addicsin participates in the regulation of EAAC1-mediated glutamate uptake (Akiduki & Ikemoto, 2008) and ER protein trafficking Ruggiero et al., 2008). We discuss these latter two physiological functions in detail (Section 2).

Arl6ip1
The "ADP-ribosylation factor-like 6 interacting protein 1 (Arl6ip1)" is the new name assigned to three independently described factors: the original Arl6ip, apoptotic regulator in the membrane of the ER (ARMER), and protein KIAA0069. The Arl6ip1 protein was first identified by yeast two-hybrid screening using mouse ARL6 as bait (Ingley et al., 1999) and as a negative regulatory factor during myeloid differentiation by differential display (Pettersson et al., 2000). Moreover, a novel protein, designated ARMER, initially discovered as a false-positive clone by yeast two-hybrid screening using Bcl-xL as bait, is also Arl6ip1 (Lui et al., 2003). In addition, Arl6ip1 has more than 96% homology with the human protein KIAA0069, the product of a cDNA isolated from the human myeloblast cell line KG-1 during a systematic effort to characterize complete cDNAs (Nomura et al., 1994). Amino acid analysis of Arl6ip1 demonstrates that it is composed of 203 amino acids and encodes a 23-kDa protein with four putative transmembrane segments (Pettersson et al., 2000). Several studies indicate that Arl6ip1 is an integral membrane protein localized to the ER (Lui et al., 2003;Pettersson et al., 2000). Furthermore, computational analysis of the topology of Arl6ip1 demonstrates that the N-and C-terminal ends are both exposed to the cytoplasm (Lui et al., 2003). Consistent with these results, Arl6ip1 has two putative casein kinase II phosphorylation motifs (amino acids 18-21 and 128-131), three putative PKC phosphorylation motifs (amino acids 94-96, 115-117, and 128-130), a N-glycosylation motif (amino acids 6-9), a prenyl group-binding motif (amino acids 72-75), and an ER retention signal in the C-terminal cytoplasmic region (amino acids 200-203) (Akiduki & Ikemoto, 2008;Lui et al., 2003) (Fig. 1). Thus, Arl6ip1 function may be controlled by diverse intracellular cell signals, but it is unknown whether these motifs are physiologically functional.
The functions of Arl6ip1 remain largely unknown, but culture studies have provided several intriguing possibilities. For example, Arl6ip1 protects HT1080 fibrosarcoma cells from apoptosis induced by serum starvation, doxorubicin, UV irradiation, tumor necrosis factor , and ER stressors by inhibiting caspase-9 activity (Lui et al., 2003). In addition, Arl6ip1 suppresses cisplatin-induced apoptosis in CaSki human cervical cancer cells by regulating the expression of apoptosis-related proteins caspase-3, caspase-9, p53, NF-B, MAPK, Bcl-2, Bcl-xL, and Bax (Guo et al., 2010a). Furthermore, Arl6ip1 is involved in cell growth, cell cycle progression, and invasion of cancer cells. Downregulation of Arl6ip1 suppresses cell proliferation and colony formation, arrests cell cycling at the G0/G1 phase, and inhibits migration of CaSki human cervical cancer cells (Guo et al., 2010b). Most relevant to the present discussion, Arl6ip1 is involved in the regulation of EAAC1. Recently, we demonstrated that Arl6ip1 is a novel addicsin-associating factor that indirectly promotes PKC-dependent EAAC1-mediated glutamate uptake by decreasing the number of addicsin molecules available for suppression of EAAC1 (Akiduki & Ikemoto, 2008).

Regulation of EAAC1 function by addicsin
The mechanisms by which addicsin regulates EAAC1 activity have not been definitively established. However, the discovery of addicsin/GTRAP3-18 has contributed greatly to our understanding of EAAC1 function. Recent evidence demonstrates two major mechanisms of addicsin-mediated regulation of EAAC1 activity. One regulatory pathway is dependent on the dynamic competition for free addicsin molecules by other addicsin molecules to form the homocomplex and by Arl6ip1 to form a heterocomplex. This addicsin-Arl6ip1 complex sequesters addicsin molecules and blocks the interaction of addicsin with EAAC1 in the plasma membrane, thereby reducing the inhibitory effect of addicsin on EAAC1-mediated glutamate uptake (Akiduki & Ikemoto, 2008;Lin et al., 2001) (Fig. 3). Second, addicsin functions as a negative regulator of EAAC1 trafficking through the ER and inhibits the cell surface expression of EAAC1 Ruggiero et al., 2008). In this section, we discuss these two mechanisms in detail.

Modulation of EAAC1-mediated glutamate uptake by addicsin
As an introduction to addicsin/GTRAP3-18-mediated regulation of EAAC1 activity, we discuss two early papers in detail. Lin et al. demonstrated that addicsin/GTRAP3-18 binds to EAAC1 and inhibits EAAC1-mediated glutamate uptake by this direct interaction (Lin et al., 2001). The second is our study showing that addicsin inhibits EAAC1-mediated glutamate uptake in a PKC activity-dependent manner while Arl6ip1 promotes glutamate uptake (also in a PKC activity-dependent manner) by inhibiting the interaction of addicsin with EAAC1 (Akiduki & Ikemoto, 2008). Lin et al. first identified addicsin/GTRAP3-18 as an EAAC1-interacting protein by yeast two-hybrid screening of a rat brain cDNA library. To evaluate whether addicsin/GTRAP3-18 modulates EAAC1 function, they examined the effect of increasing addicsin/GTRAP3-18 expression on EAAC1-mediated glutamate uptake in vitro and in vivo. First, they showed that glutamate uptake decreased progressively with increasing expression of addicsin/GTRAP3-18 in HEK293 cells. Subsequent kinetic analyses in HEK293, C6BU-1, and COS7 cells revealed that elevated expression of addicsin decreased the glutamate affinity of EAAC1 without altering the maximal transport velocity (correlated with expression). Furthermore, HEK293 cells coexpressing addicsin/GTRAP3-18 and a truncated EAAC1 missing the addicsin/GTRAP3-18 association region showed higher glutamate uptake than cells expressing wild-type EAAC1. In addition, this truncated EAAC1 had a higher affinity for glutamate, suggesting that addicsin/GTRAP3-18 normally reduces EAAC1-mediated glutamate uptake by binding to this association region and reducing transporter glutamate affinity. Next, they evaluated the effect of intraventricular injection of an addicsin/GTRAP3-18 antisense mRNA on EAAC1-mediated glutamate uptake in vivo. The antisense treatment resulted in reduced addicsin/GTRAP3-18 expression, a significant increase in cortical EAAC1-mediated glutamate uptake, and an increase in glutamate affinity compared to saline-treated or sense mRNA-treated control animals. In conclusion, addicsin/GTRAP3-18 can negatively modulate EAAC1-mediated glutamate uptake by a direct interaction with EAAC1.
We first isolated addicsin as a novel protein richly expressed in the amygdala of mice under chronic morphine treatment. Addicsin has a tendency to form the multimeric complex in vitro (Ikemoto et al., 2002;Lin et al., 2001). The initial discovery of addicsin prompted us to perform yeast two-hybrid screening of an amygdala cDNA library constructed from chronic morphine-administered mice. From this screen, we identified Arl6ip1 as a candidate addicsin-interacting protein. As described in section 1.3, Arl6ip1 is an anti-apoptotic protein located in the ER. As previously described, addicsin inhibits EAAC1-mediated glutamate uptake by direct association at the plasma membrane (Lin et al., 2001), so we speculated that Arl6ip1 upregulates EAAC1-mediated glutamate transport by inhibiting the interaction between addicsin and EAAC1 (Fig. 3).
As a first step to verify this hypothesis, we investigated whether addicsin could bind Arl6ip1 in vitro and in vivo. To eliminate the possibility of false-positive clones, reconfirmation tests using a full length mouse Arl6ip1 as prey or bait were performed. This tests revealed the specific interaction with addicsin in the yeast AH109 strain. We next examined the reproducibility of this screening result by yeast two-hybrid screening using a different cDNA library prepared from whole brains of 7-week-old mice. We obtained 20 positive clones that clearly displayed -galactosidase activity (the gene driven by the protein-protein interaction in the two-hybrid screen). Among these positive clones, 11 were identical to Arl6ip1 cDNA (M.J. Ikemoto et al., unpublished data), confirming the interaction with addicsin and Arl6ip1 in the yeast AH109 strain. We then performed immunoprecipitation analysis, glycerol gradient analysis, and immunocytochemical analysis to directly test the interaction between Arl6ip1 and addicsin in vitro. For this purpose, we prepared cell lysates from NG108-15 cells expressing FLAG-tagged Arl6ip1 (Arl6ip1-FLAG), Myc-tagged addicsin (addicsin-myc), or both. Immunoprecipitation analysis of these cell lysates demonstrated that Arl6ip1-FLAG specifically interacted with addicsin-myc in the cell lysates prepared from coexpressing cells, but not from cells expressing Arl6ip1-FLAG or addicsin-myc alone. Glycerol gradient analysis revealed that the elution profile of Arl6ip1-FLAG was similar to that of addicsin-myc. The elution peaks of both proteins were observed in the fraction with a deduced molecular mass of 24 kDa. Moreover, the elution peak of the addicsin homodimer was present in the 44-kDa fraction, suggesting that addicsin forms Arl6ip1-addicsin heterodimers and addicsin-addicsin homodimers in vitro. Immunocytochemical analysis in NG108-15 cells overexpressing Arl6ip1-FLAG and addicsin-myc demonstrated subcellular colocalization (M.J. Ikemoto et al., unpublished data). To examine the interaction of both proteins in vivo, we performed in vivo immunoprecipitation assays of whole brain lysates using an anti-Arl6ip1 polyclonal antibody (generated from a synthetic peptide spanning amino acids 185-199 of mouse Arl6ip1) that again revealed a specific interaction between Arl6ip1 and addicsin. Western blot analysis demonstrated that Arl6ip1 was widely expressed in the mature brain and showed substantial regional overlap with addicsin. In addition, immunohistochemical staining confirmed that Arl6ip1 was widely expressed in the mature brain and localized in neuron-like cells. The neural expression pattern of Arl6ip1 was the same as addicsin, suggesting that Arl6ip1 is colocalized with addicsin in the mature CNS. We concluded that addicsin specifically interacted with Arl6ip1 in vitro and in vivo.
As a second step, we then determined the Arl6ip1-and addicsin-binding regions on addicsin. If Arl6ip1 does regulate EAAC1 activity by competitively binding to addicsin molecules and thus preventing the formation of addicsin homodimers that downregulate EAAC1 activity, the Arl6ip1-and addicsin-binding regions on addicsin should be located close enough for such a competitive interaction. Immunoprecipitation assays using several addicsin truncation mutants indicated that Arl6ip1 associated with full length addicsin (wt), a truncation lacking the C-terminal region at amino acids 145-188 (d1), a deletion mutant of the N-terminal domain at amino acids 1-102 (d2), and a mutant missing the region containing the C-terminal phosphorylation motif at amino acids 136-144 (d3). However, Arl6ip1 could not interact with a mutant lacking a portion of the hydrophobic region at amino acids 103-117 (d4). As expected, addicsin was able to associate with the wt, d1, d2, or d3 mutant, but not the d4 truncation mutant, indicating that the hydrophobic region at amino acids 103-117 of addicsin is a crucial domain for the formation of addicsin-addicsin homodimers and addicsin-Arl6ip1 heterodimers (Fig. 1). These results strongly support our hypothesis that Arl6ip1 antagonizes addicsin-mediated downregulation of EAAC1 activity by sequestering free addicsin.
As a third step, we investigated whether Arl6ip1 had a positive effect on EAAC1-mediated glutamate uptake. For this purpose, we selected C6BU-1 glioma cells that expressed EAAC1 as the principal or only EAAT (Palos et al., 1996). We created two stably expressing C6BU-1 cell lines, designated C6BU-1-pSw-addicsin and C6BU-1-pSw-Arl6ip1. In these cell lines, we could strictly control the expression levels of V5-tagged addicsin (addicsin-V5) or V5-tagged Arl6ip1 (Arl6ip1-V5) by exposure to 10 nM mifepristone (11 -[4-dimethylamino]phenyl-17 -hydroxy-17-[1-propynyl]estra-4,9-dien-3-one), a synthetic 19-norsteroid. In addition, a cell viability assay demonstrated that upregulation of Arl6ip1-V5 or addicsin-V5 by exposure to 10 nM mifepristone was not cytotoxic, making these cell lines excellent models to evaluate the effects of changing Arl6ip1 and addicsin expression on the functional activity of EAAC1. Compared to control cells untreated with mifepristone or the PKC agonist PMA, the upregulation of Arl6ip-V5 or addicsin-V5 by 10 nM mifepristone alone did not change EAAC1-mediated glutamate uptake. When these cells were stimulated with 100 nM PMA alone, the glutamate uptake activity in C6BU-1-pSw-addicsin cells and C6BU-1-pSw-Arl6ip1 cells increased about two-fold compared to untreated controls. EAAC1-mediated glutamate uptake was significantly lower in C6BU-1-pSw-addicsin cells stimulated with both mifepristone and PMA compared to C6BU-1-pSw-addicsin cells treated with PMA alone, indicating that activation of addicsin expression inhibited PKC-dependent EAAC1 activity. In contrast, C6BU-1-pSw-Arl6ip1 cells treated with PMA and mifepristone exhibited a threefold increase in glutamate uptake compared to the same line treated with PMA alone, indicating that Arl6ip1 overexpression enhanced PKC-dependent EAAC1 activity. On the other hand, the nonstimulating PMA analog 4 phorbol did not increase glutamate uptake relative to controls.
To further support these conclusions, we performed a knockdown experiment by transient transfection of double-stranded siRNAs into C6BU-1-pSw-Arl6ip1 cells to investigate the effect of decreased addicsin expression on EAAC1-mediated glutamate uptake. As expected, cells transfected with either of two alternative addicsin siRNAs showed about a two-fold increase in glutamate uptake in response to PMA exposure compared to cells treated with control scrambled siRNA. The elevated glutamate uptake concomitant with addicsin knockdown strongly supported the proposed mechanism for EAAC1 regulation by addicsin and Arl6ip1.
As a fourth step, we then examined the subcellular localization of Arl6ip1 in C6BU-1-pSw-Arl6ip1 cells. Western blot analysis revealed that Arl6ip1-V5 expression levels were unaffected by 100 nM PMA exposure. Immunocytochemical analysis demonstrated that Arl6ip1-V5 was predominantly localized to cytoplasmic structures such as the ER and that this subcellular expression pattern was not changed by PMA. Furthermore, cell biotinylation analysis indicated that Arl6ip1 did not interact with the plasma membrane, consistent with our previous result that Arl6ip1 failed to interact with EAAC1 by immunoprecipitation. Therefore, Arl6ip1 was localized to the ER under all conditions tested and acted to "trap" addicsin molecules in Arl6ip1-addicsin heterodimers, thus preventing the direct interaction of addicsin with EAAC1. To confirm our hypothesis, we produced an addicsin mutant that lacked interaction with Arl6ip1 but not with other addicsin molecules. Fine mutational analysis was used to separate the Arl6ip1-and addicsin-binding regions within the addicsin d4 region. We compared addicsin sequences among various species and noted that two amino acids at positions 110 and 112 of mouse addicsin were completely conserved from fruit fly to human. We created a double-mutated form of addicsin that substituted both the native tyrosine at amino acid 110 and the leucine at amino acid 112 with alanine. The mutant, designated addicsin Y110A/L112A (or addicsinYL), showed markedly less binding to Arl6ip1 (40% of wild-type addicsin) but normal wild-type binding to addicsin, as revealed by immunoprecipitation. In addition, a cell biotinylation assay indicated that addicsinYL was unable to localize to the plasma membrane, suggesting that addicsinYL lost EAAC1-binding activity. To evaluate the effect of addicsinYL on EAAC1-mediated glutamate uptake, we created a conditional C6BU-1 cell line, designated C6BU-1-pSw-addicsinYL. This cell line exhibited mifepristone-dependent upregulation of V5-tagged addicsinYL and increased glutamate uptake in response to PMA that was unchanged by mifepristone-induced upregulation of addicsinYL. That is, glutamate uptake was not reduced by induced addicsinYL expression. These data strongly suggest that addicsin is a key negative regulator of EAAC1 in the plasma membrane and that Arl6ip1 is a negative regulator of addicsin.
As a final step, we examined the effect of addicsin PKC phosphorylation sites on EAAC1mediated glutamate uptake in C6BU-1 cells. Addicsin has putative PKC phosphorylation motifs at amino acids 18-20 and 138-140, and PKC activation increases EAAC1-mediated glutamate uptake. We established conditional C6BU-1 cell lines, designated C6BU-1-pSw-addicsinS18A and C6BU-1-pSw-addicsinS138A. C6BU-1-pSw-addicsinS18A cells expressed a V5-tagged addicsin point mutant that substituted native serine 18 for alanine in the Nterminal motif in response to mifepristone, while C6BU-1-pSw-addicsinS138A cells expressed a V5-tagged addicsin point mutant that substituted native serine 138 for alanine in the C-terminal motif. These cells showed no cytotoxicity in response to 10 nM mifepristone. In contrast to cells expressing wild-type addicsin, expression of addicsinS18A did not suppress the PMA-induced increase in EAAC1-mediated glutamate uptake. Moreover, increased expression of addicsinS18A caused a significant increase in glutamate uptake even without PMA stimulation by a dominant negative effect. Similarly, addicsinS138A expression did not suppress the PMA-induced increase in EAAC1-mediated glutamate uptake. Thus, these mutations abolished the inhibitory effect of addicsin. However, in contrast to addicsinS18A, addicsinS138A expression had no influence on EAAC1-mediated glutamate uptake activity in the absence of PMA stimulation. Both serine 18 and serine 138 within the putative PKC phosphorylation motifs are critical for the negative regulation of EAAC1-mediated glutamate uptake and suggest that the PKC phosphorylation site at serine 138 is functional under physiological conditions. Based on these data, we proposed the regulatory model of EAAC1-mediated glutamate uptake illustrated in Fig. 3. If addicsin expression is high enough relative to Arl6ip1 to form many more addicsin homodimers than addicsin-Arl6ip1 heterodimers, EAAC1-mediated glutamate uptake is reduced. Furthermore, activation of the PKC isozyme that phosphorylates addicsin at S18 or S138 may further potentiate this negative regulation. On the other hand, if addicsin expression is low enough or Arl6ip1 expression high enough that formation of heterodimers predominates, fewer addicsin homodimers are available to suppress EAAC1 activity. The resulting decrease in addicsin-EAAC1 binding will enhance the catalytic efficacy of EAAC1, in a PKC-activity dependent manner. In sum, Arl6ip1 acts as a positive regulator of EAAC1-mediated glutamate uptake (Fig. 3) and may therefore possess significant neuroprotective efficacy against neurodegenerative diseases linked to excitotoxicity and oxidative stress.

Modulation of ER protein trafficking by addicsin
Addicsin is a member of the PRAF protein family with homology to PRA1 and PRAF2 (JM4) (Schweneker et al., 2005). PRA1 is associated with the Golgi membrane and interacts with Rab, a member of the Ras superfamily of small GTP-binding proteins, which regulates intracellular protein trafficking (Bucci et al., 1999;Liang & Li, 2000;Martincic et al., 1997).

www.intechopen.com
Immunocytochemical studies reveal that mature addicsin is present in both the plasma membrane and the intracellular compartment, including the ER (Ikemoto et al., 2002;Watabe et al., 2007Watabe et al., , 2008. Thus, addicsin may also be involved in intracellular protein trafficking. To investigate this possibility, we examined EAAC1 oligosaccharide residues under conditions of varying addicsin expression. The oligosaccharide residues on EAAC1 are an excellent indicator of the extent of ER-to-Golgi trafficking and plasma membrane localization because the newly synthesized EAAC1 is N-glycosylated with high mannose oligosaccharide chains that are subsequently processed into more complex sugar chains by resident Golgi enzymes (Yang & Kilberg, 2002). In HEK293T cells coexpressing addicsin, EAAC1 is predominantly modified by high mannose oligosaccharides, suggesting that EAAC1 proteins are largely confined to the ER. Furthermore, addicsin delays oligosaccharide maturation of EAAC1 but does not induce EAAC1 degradation . These data suggest that addicsin delays ER-to-Golgi trafficking of EAAC1. Moreover, addicsin inhibits ER-to-Golgi trafficking of dopamine transporter, GABA transporter 1, and several G-protein-coupled receptors, including 2 -adrenergic receptor, 1receptor, and D 2 receptor . Furthermore, addicsin inhibits the function of RTN2B, a member of the reticulon protein family localized in the ER, which enhances ER-to-Golgi trafficking of EAAC1 . As addicsin, RTN2B, and EAAC1 are coexpressed in neurons, they may interact in one complex. Indeed, addicsin and EAAC1 can interact with RTN2B by binding to different regions of the protein. In addition, coexpression of RTN2B and EAAC1 in HEK293 cells increases EAAC1 cell surface expression, while increasing addicsin expression blocks this effect. Thus, EAAC1 trafficking is inhibited by addicsin and facilitated by RTN2B . Based on these data, Liu et al. proposed a model in which the regulation of ER trafficking governs the activity and density of EAAC1 at the plasma membrane. Under normal conditions, RTN2B facilitates EAAC1 trafficking from the ER because basal expression of addicsin is too low to have an inhibitory effect. Under stressful conditions, such as oxidative and chemical stress, addicsin expression is upregulated and the inhibitory effect on EAAC1 trafficking predominates over the facilitating effect of RTN2B . Addicsin can delay ER-to-Golgi trafficking of structurally and functionally distinct proteins in addition to EAAC1. Thus, addicsin is a stress-induced multifunctional protein that participates in various physiological and pathological functions by regulating ER trafficking of many membrane effector proteins, including receptors and transporters.

Addicsin & neurological disorders
Recent studies have also linked addicsin to the pathophysiology of several neurological diseases, including drug addiction, schizophrenia, and epilepsy. In this section, we focus on these diseases and review the putative pathophysiological functions of addicsin in the mammalian CNS.

Drug abuse
Several studies demonstrate that addicsin is involved in drug abuse, the development of morphine dependence (Ikemoto et al., 2002;Wu et al., 2011), and ethanol tolerance (C. Li et al., 2008). In an effort to clarify the molecular mechanism of opiate addiction, we performed subtractive hybridization of mRNA expressed in the amygdala of mice treated with repeated doses of morphine and identified addicsin mRNA as a factor selectively upregulated relative to drug-naïve mice (Ikemoto et al., 2000(Ikemoto et al., , 2002. Upregulation of addicsin mRNA was specifically induced by chronic, but not acute, morphine administration and was completely inhibited by coadministration of naloxone, an opiate r e c e p t o r a n t a g o n i s t ( I k e m o t o e t a l . , 2 0 0 2 ) . I n t h a t s t u d y , w e u s e d a m o r p h i n e administration protocol that had been previously shown to induce morphine dependence and tolerance (Kaneto et al., 1973). Thus, our data strongly suggested that addicsin was involved in the development of morphine dependence in this animal model. Later reports have confirmed our findings by directly demonstrating that addicsin is directly involved in the development of morphine dependence (Wu et al., 2011). Chronic morphine treatment upregulated addicsin in prefrontal cortex, nucleus accumbens, and amygdala, which are regions known to be critical for the development of morphine dependence and other addictive behaviors. Furthermore, addicsin knockdown by infusion of addicsin antisense nucleotides into the cerebral ventricles significantly decreased withdrawal behaviors following chronic morphine treatment in rats (Wu et al., 2011). Addicsin knockdown suppressed the upregulation of opioid receptors, the activation of the dopamine-and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), and MAPK activation normally induced by chronic morphine treatment. Furthermore, addicsin knockdown enhanced the degradation of opioid receptors through the ubiquitinproteasome pathway (Wu et al., 2011). These data suggest that addicsin directly contributes to the regulation of opioid receptor stability and the development of morphine dependence by suppressing opioid receptor expression and the activation of DARPP-32 and MAPK. The opioid receptor knockout mice do not develop analgesic tolerance to morphine without affecting the development of physical dependence (Kieffer & Gaveriaux-Ruff, 2002;Nitsche et al., 2002;Y. Zhu et al., 1999). Thus, further investigations are needed to clarify whether addicsin is involved in analgesic tolerance.
Ethanol-induced cellular responses are analogous to those elicited by heat shock stresses (Piper, 1995;Wilke et al., 1994). Similarly, addicsin expression is enhanced in response to various environmental stressors, such as oxidative stress and heat shock stress (R. . Furthermore, our study demonstrated that addicsin plays an important role in the development of morphine dependence and tolerance (Ikemoto et al., 2002). In the light of these observations, addicsin is considered to be essential for the development of ethanol tolerance. To address this issue, addicsin knockdown flies were generated. To estimate ethanol tolerance objectively, the inebriation test was performed (Bellen, 1998). Flies were exposed to ethanol vapor, and the mean elution time (MET) was measured three times after inebriation. The addicsin knockdown flies showed no difference between the first MET and third MET, while wild-type flies exhibited a significant higher third MET (C. Li, et al., 2008), indicating that addicsin knockdown flies failed to acquire ethanol tolerance.

Schizophrenia
Glutamatergic neurotransmission and plasticity are disrupted in patients with schizophrenia (Javitt, 2010;Kantrowitz & Javitt, 2010;Paz et al., 2008). This has led some researchers to speculate that EAATs and EAAT-interacting proteins that regulate glutamate transport efficacy or transporter expression may be abnormal in patients with schizophrenia (Bauer et al., 2008;Huerta et al., 2006). Indeed, addicsin/JWA transcripts were overexpressed in the thalamus (Huerta et al., 2006) and the anterior cingulate cortex of schizophrenics as shown by in situ hybridization (Bauer et al., 2008). In these studies, the protein expression levels of addicsin/JWA were not determined. In addition, expression of EAAT3, the human homolog of EAAC1, was also upregulated in the anterior cingulate cortex of schizophrenic patients (Bauer et al., 2008). Furthermore, a microarray study of multiple human brain regions demonstrates that the anterior cingulate cortex is more vulnerable to these aberrant gene expression patterns (Katsel et al., 2005), and hypofrontality is a key feature of schizophrenia. Addicsin is thus a promising target for further research focusing on the role of glutamate transporters in schizophrenia. Moreover, addicsin regulates trafficking of a plethora of other membrane proteins, including dopamine receptors, suggesting another pathway through which addicsin participates in the pathogenesis of schizophrenia.

Epilepsy
Anatomical analysis of EAAT expression reveals that EAAC1 is enriched in neurons and particularly localized to inhibitory GABAergic neurons (Conti et al., 1998;He et al., 2000;Rothstein et al., 1994). Cerebroventricular injection of EAAC1 antisense oligonucleotides caused no elevation of extracellular glutamate in the rat striatum but did produce mild neurotoxicity and epileptiform activity (Rothstein et al., 1996). Furthermore, epilepsy in EAAC1 knockdown rats is caused by decreased GABA synthesis (Sepkuty et al., 2002). Glutamate is a precursor for GABA synthesis, so molecules that alter the intracellular availability of glutamate in GABAergic interneurons, including addicsin/GTRAP3-18, may have an important role in epileptogenesis or ictogenesis. In a recent study of the antiepileptic drug levetiracetam (LEV), changes in the expression of addicsin/GTRAP3-18, glutamate transporters, and GABA transporters were examined in a rat post-traumatic epilepsy model induced by FeCl 3 injection into the amygdala. Administration of LEV increased expression of EAAC1 and GABA transporter 3 (GAT-3) but decreased expression of addicsin/GTRAP3-18 in the rat hippocampal formation (Ueda et al., 2007). These results suggest that both the suppression of glutamatergic excitation and the enhancement of GABAergic inhibition induced by chronic LEV administration are due to the upregulation of EAAC1 and GAT-3 subsequent to downregulation of addicsin/GTRAP3-18. A long-lasting suppression of addicsin/GTRAP3-18 expression was observed in the rat pentylenetetrazole (PTZ)-induced kindling model of epilepsy (Ueda et al., 2006). Similarly, antisense-mediated knockdown of addicsin/GTRAP3-18 decreases seizure threshold and promotes PTZ kindling. In addition, addicsin/GTRAP3-18 knockdown increases basal release of glutamate and GABA in the rat hippocampal formation, indicating that knockdown of addicsin/GTRAP3-18 promotes GABA synthesis (Ueda et al., 2006). These studies, demonstrating that addicsin can increase GABA synthesis by increasing the substrate (i.e., glutamate) supply, define addicsin as a novel therapeutic target in epilepsy.

Other neurological disorders
Addicsin directly modulates glutamate and cysteine uptake by EAAC1, suggesting that addicsin participates in the pathogenesis of neurological disorders associated with excitotoxicity and oxidative stress. Here we briefly discuss some representative EAAC1 functions relevant to CNS pathology. A recent study demonstrated that EAAC1-deficient mice developed age-dependent brain atrophy and behavioral abnormalities in the cognitive and motivational domains. In addition, EAAC1 knockout mice displayed impaired GSH homeostasis and age-dependent neurodegeneration, and these pathologies were rescued by treatment with the membrane permeable cysteine precursor N-acetylcysteine (Aoyama et al., 2006). These EAAC1 knockout mice also display dicarboxylic aminoaciduria and significant motor impairments (Peghini et al., 1997). These results indicate that EAAC1 functions as a cysteine transporter in neurons and sustains intracellular GSH to ameliorate oxidative stress in vivo. Furthermore, neuronal glutamate uptake can also regulate memory formation (Levenson et al., 2000;Maleszka et al., 2000). The increase of EAAC1-mediated neuronal glutamate uptake is associated with the induction and expression of early phase long-term potentiation (LTP) in the CA1 area of the hippocampal formation and with contextual fear conditioning, a form of hippocampus-dependent memory thought to depend on induction of LTP (Levenson et al., 2002). These results suggest that regulation of glutamate uptake by EAAC1 is a physiologically important mechanism for the modulation of synaptic strength during long-term changes in synaptic efficacy (plasticity). Thus, dysfunction of EAAC1 induced by aberrant addicsin expression may lead to neurodegeneration and cognitive decline. Of particular interest is the role of addicsin in the pathogenesis of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. These questions warrant further research.

Future research perspective
Despite these advances, our understanding of the regulatory mechanisms of addicsin expression and the range of addicsin functions is far from complete. The elucidation of the regulatory mechanism of addicsin expression under basal and pathological conditions is essential for understanding the physiological and pathological roles of addicsin. For instance, while addicsin has consensus PKC phosphorylation sequences, it is unclear whether PKC actually phosphorylates addicsin and controls addicsin functions in vivo. It is also unknown whether or how PKC phosphorylation affects the interaction between addicsin and Arl6ip1. To overcome these challenges, it is crucial to clarify whether PKC phosphorylation sites of addicsin are physiologically controlled by PKC signaling and by which PKC isoforms. Furthermore, it remains controversial whether addicsin is an integral membrane protein. Our results strongly support the notion that addicsin is a membrane-associating protein with a soluble and membrane-localized form. Thus, it is important to clarify the different molecular features and functions of the soluble and membrane-localized forms of addicsin. Second, in vivo functional studies are still needed to clarify the physiological and pathological functions of addicsin. Accumulating evidence suggests that addicsin participates in various physiological and pathological processes in vivo, but the molecular mechanisms controlling the selective interaction of addicsin with multiple targets, including receptors and transporters, are unknown. Furthermore, many reports demonstrate that the physiological and pathological roles of addicsin are observed when expression of addicsin is increased by various stresses, including oxidative and chemical stress. Thus, the production of animal models that overexpressed addicsin in a tissue-or region-specific manner may be useful to analyze addicsin functions in various tissues, including the brain. At present, no studies have been undertaken in tissues outside the brain, although addicsin is ubiquitously expressed in kidney, heart, and liver (Butchbach et al., 2002;Ikemoto et al., 2002).
We believe that studies using transgenic or conditional knockin/knockout animal models will lead to novel insights into addicsin function. Of particular interest is whether dysfunctional addicsin expression or function can lead to neurodegenerative diseases through dysregulation of EAAC1 or other proteins. Finally, we hope that studies on addicsin will continue to advance our understanding of the role of addicsin in the pathogenesis of diseases, such as drug abuse, and lead to the development of curative therapies.

Conclusion
In this chapter, we argued that Arl6ip1 is a novel addicsin-interacting protein that indirectly promotes PKC-dependent, EAAC1-mediated glutamate uptake by inhibiting the interaction of addicsin with EAAC1 at the plasma membrane. Based on these findings, we proposed the regulatory model of EAAC1-mediated glutamate uptake illustrated in Fig. 3. In this model, EAAC1-mediated glutamate uptake activity can be negatively and positively regulated by PKC activity depending on dynamic modulation by addicsin complexes. Thus, the cellular dynamics of addicsin is a key element regulating EAAC1-mediated glutamate uptake. The study of addicsin is still in its infancy, but future findings on the physiological and pathophysiological functions of addicsin could greatly clarify the role of EAAC1 (and other proteins regulated by addicsin) in health and disease.

Acknowledgment
This work was supported by a research grant from the National Institute of Advanced Industrial Science and Technology (AIST) of Japan. We thank Dr. S. Akiduki, M.Sc. M. Takumori, Dr. M. Ohtomi, Dr. K. Inoue, and Dr. T. Ochiishi for helpful discussions, and Ms. K. Nemoto for her excellent technical assistance.