**3. 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.

#### **3.1 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

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., 2007, 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 (Ruggiero et al., 2008). 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 β2-adrenergic receptor, α1 β receptor, and D2 receptor (Ruggiero et al., 2008). 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 (Liu et al., 2008). 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 (Liu et al., 2008). 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 (Liu et al., 2008). 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,

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

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

including receptors and transporters.

mammalian CNS.

**3.1 Drug abuse** 

**3. Addicsin & neurological disorders** 

with repeated doses of morphine and identified *addicsin* mRNA as a factor selectively upregulated relative to drug-naïve mice (Ikemoto et al., 2000, 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 receptor antagonist (Ikemoto et al., 2002). In that study, we used a morphine 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 δ 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 δ opioid receptors through the ubiquitin– proteasome pathway (Wu et al., 2011). These data suggest that addicsin directly contributes to the regulation of δ opioid receptor stability and the development of morphine dependence by suppressing δ opioid receptor expression and the activation of DARPP-32 and MAPK. The δ 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. Chen et al., 2007). 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.

#### **3.2 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.

#### **3.3 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 FeCl3 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.

#### **3.4 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.

354 Biochemistry

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

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 FeCl3 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.

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

pathogenesis of schizophrenia.

**3.4 Other neurological disorders** 

**3.3 Epilepsy** 

Fig. 1. A schematic presentation of addicsin and Arl6ip1

### **4. 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.

Fig. 2. A scheme of the proposed physiological functions 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.
