Pharmacology, Biochemistry and Behavior 127 (2014) 70–81

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GLT-1 transporter: An effective pharmacological target for various neurological disorders Neha Soni, B.V.K. Reddy, Puneet Kumar ⁎ Department of Pharmacology, ISF College of Pharmacy, Moga 142001, Punjab, India

a r t i c l e

i n f o

Article history: Received 17 April 2014 Received in revised form 1 September 2014 Accepted 3 October 2014 Available online 13 October 2014 Keywords: Glutamate Excitotoxicity GLT-1 Neurological disorders

a b s t r a c t L-Glutamate is the predominant excitatory neurotransmitter in the central nervous system (CNS) and is directly and indirectly involved in a variety of brain functions. Glutamate is released in the synaptic cleft at a particular concentration that further activates the various glutaminergic receptors. This concentration of glutamate in the synapse is maintained by either glutamine synthetase or excitatory amino acid proteins which reuptake the excessive glutamate from the synapse and named as excitatory amino acid transporters (EAATs). Out of all the subtypes GLT-1 (glutamate transporter 1) is abundantly distributed in the CNS. Down-regulation of GLT-1 is reported in various neurological diseases such as, epilepsy, stroke, Alzheimer's disease and movement disorders. Therefore, positive modulators of GLT-1 which up-regulate the GLT-1 expression can serve as a potential target for the treatment of neurological disorders. GLT-1 translational activators such as ceftriaxone are found to have significant protective effects in ALS and epilepsy animal models, suggesting that this translational activation approach works well in rodents and that these compounds are worth further pursuit for various neurological disorders. This drug is currently in human clinical trials for ALS. In addition, a thorough understanding of the mechanisms underlying translational regulation of GLT-1, such as identifying the molecular targets of the compounds, signaling pathways involved in the regulation, and translational activation processes, is very important for this novel drug-development effort. This review mainly emphasizes the role of glutamate and its transporter, GLT-1 subtype in excitotoxicity. Further, recent reports on GLT-1 transporters for the treatment of various neurological diseases, including a summary of the presumed physiologic mechanisms behind the pharmacology of these disorders are also explained. © 2014 Elsevier Inc. All rights reserved.

1. Introduction The transmembrane transport of neurotransmitters is of fundamental importance for proper signaling between neurons (Ribeiro and Patocka, 2013; Gether et al., 2006). The transport processes are mediated by distinct classes of membrane transport protein that have key roles in controlling the neurotransmitter concentration in the synaptic cleft, as having more active reuptake phenomenon not only helps to replenish the pool of releasable transmitter but may also reduce the extent and duration of signaling to the postsynaptic cell (Eulenburg and Gomeza, 2010). Contrarily, loss of reuptake increases the activation of receptors but results in the depletion of stores (Blakely and Edwards, 2012). Therefore, it is clear that any imbalance between the release and reuptake of neurotransmitter may lead to disturbance in the neuronal

⁎ Corresponding author at: Pharmacology Division, ISF College of Pharmacy, Moga 142001, Punjab, India. Tel.: +91 1636 324200, 324201; fax: +91 1636 239515. E-mail address: [email protected] (P. Kumar).

http://dx.doi.org/10.1016/j.pbb.2014.10.001 0091-3057/© 2014 Elsevier Inc. All rights reserved.

signaling between the neurons which may lead to abnormalities and central nervous system (CNS) disorders. L-Glutamate is the predominant excitatory neurotransmitter in the CNS and is directly and indirectly involved in a wide variety of brain functions. It plays a very important role in the pathogenesis of many neurological disorders such as epilepsy, ALS (amyotrophic lateral sclerosis), cerebral ischemia, schizophrenia, parkinsonism, and Alzheimer's disease (Massie et al., 2010; Nakagawa and Kaneko, 2013; Annweiler et al., 2014; Kleteckova et al., 2014). Glutamatergic neuron stimulation will cause the release of glutamate into the synapse where its concentration will transiently rise to low millimolar concentrations it will activate ionotropic and metabotropic glutamate receptors (Rusakov et al., 2011; Shan et al., 2012). Excessive glutamate receptor stimulation is toxic to neurons, and glutamate transporters rapidly clear glutamate from the synapse (Mehta et al., 2013). Glutamate receptors differ considerably in their kinetics of activation, and also by the glutamate concentrations to which they are exposed. Thus, glutamate transporters play the important role of regulating extracellular glutamate concentrations to maintain dynamic synaptic signaling processes (Vandenberg

N. Soni et al. / Pharmacology, Biochemistry and Behavior 127 (2014) 70–81

and Ryan, 2013). There are a number of transporter families that transport glutamate and include the plasma membrane excitatory amino acid transporters (EAATs), the vesicular glutamate transporters (VGLUTs), and the glutamate–cysteine exchanger (Anderson and Swanson, 2000). The emphasis in this review is on the role of GLT-1/ EAAT-2 as a new therapeutic target to treat various neurological disorders because out of all the transporter subtypes GLT-1/EAAT-2 is abundantly distributed in the CNS. 2. History Shashidharan et al. (1994) found that glutamate transporters are the membrane-bound proteins localized in glial cells and/or presynaptic glutamatergic nerve endings and are essential for the removal and termination of action of the excitatory neurotransmitter glutamate from the synapse (Shashidharan et al., 1994). Several subtypes of glutamate transporters had been defined by differences in sequence, pharmacology, tissue distribution, and channel-like properties by Rothstein et al. (1995). By screening a human brainstem and cerebellum cDNA library, Shashidharan et al. (1994) isolated a cDNA corresponding to the GLT-1/EAAT-2 gene, and also reported that predicted 565-amino acid protein was homologous to a rat brain sodium-dependent glutamate/aspartate transporter (Krishnan et al., 1993). After these findings many scientists start contributing to find the role of these transporters in various neurological disorders. The major historical aspects of GLT1/EAAT-2 are summarized in Table 1. 3. Glutamate transporters: classification and distribution Glutamate transporters play important roles in the termination of excitatory neurotransmission and in providing cells throughout the body with glutamate for metabolic purposes (Kanai et al., 2013). Generally, neurotransmitter transporters can be classed as intracellular vesicular transporters that are responsible for sequestering transmitters from the cytoplasm into synaptic vesicles, and plasma membrane transporters that are responsible for sequestering released transmitter from the extracellular space (Gether et al., 2006). There are three subclasses of intracellular transporter: the vesicular amine transporters (the solute carrier (SLC18) gene family), the vesicular inhibitory amino acid transporter family (SLC32) and the vesicular glutamate transporters (SLC17 gene family). There are two major subclasses of plasma membrane transporter: the high-affinity glutamate transporters (excitatory amino acid transporters) and the Na + –Cl− coupled transporters (Kanai and Hediger, 2003; N.H. Chen et al., 2004; W. Chen et al., 2004). A number of different membrane transporters have been identified that are capable of regulating the flux of L -glutamate into and out of the cells (Danbolt, 2001). These transporters are commonly differentiated based upon their ionic dependence and include both the sodium independent system and the sodium-dependent system. On the basis of stoichiometry of glutamate transporters and the prevailing ionic environment, glutamate transporter concentrates glutamate more than 10,000-fold across the cell membranes (Patel et al., 2004; Grewer et al., 2000). Because of these factors and importantly high concentrating capacity of glutamate, it is generally accepted that the majority of glutamate transport in the CNS, particularly as related to excitatory transmission, is mediated by a group of high-affinity, sodium-dependent, EAATs, thereby terminating the transmitter signal and protecting neurons from an excitotoxic action of glutamate (Bridges and Esslinger, 2005). EAATs are five subtypes (Table 2). 4. How GLT-1 is different from other subtypes Quantitative studies demonstrate that EAAT-2 density is up to 8500 molecules per μm2 in the hippocampus glia cell membrane,

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which is four times higher than glutamate aspartate transporter (GLAST) density in the hippocampus (Lehre and Danbolt, 1998). EAAT-1/GLAST immunostaining and protein expression are most prominent in the cerebellum with moderate levels in other structures such as the hippocampus and the forebrain. In contrast, GLT-1 expression is mainly found in the forebrain regions with minor expression in the cerebellum (Perego et al., 2000). The astrocyte selective glutamate transporter EAAT-2 has been shown to be paramount in keeping extracellular glutamate below excitotoxic levels (Rothstein et al., 1995). The reported Km values of GLT-1/EAAT-2 for glutamate are at around 20 μM and the affinities of GLAST/EAAT1 and EAAT3 differ from GLT-1/ EAAT-2 with a factor of b 2 (Danbolt, 2001), while the affinities of EAAT4 and EAAT5 are, respectively, one order of magnitude higher and lower. EAAT4 and EAAT5 have the largest chloride conductance, and may function more as inhibitory glutamate receptors than as transporters (Veruki et al., 2006). GLT-1/EAAT2 is found mainly responsible for maintaining glutamate homeostasis, which if disturbed due to downregulation of these transporters causes excitability which in turn leads to neuro-degeneration. Also, GLT-1/EAAT2 and GLAST/EAAT1 are the only glutamate transporters expressed in brain astrocytes as both EAAT3 (Holmseth et al., 2012) and EAAT4 (Dehnes et al., 1998; de Vivo et al., 2010) are neuronal. Within the CNS, EAAT5 is preferentially expressed in the retina and very low in the brain. On molecular basis, it was found that, the glutamate transporter GLT-1/EAAT2 is different from other subtypes in two respects. First, Li (Lithium) cannot support transport by GLT-1/EAAT2, whereas it can support transport by the other EAATs, and second, GLT-1/EAAT2 is sensitive to a wider range of blockers than other subtypes (Hughes, 2009). 5. Mechanism involved in GLT-1 mediated glutamate transport Glutamate transporters play important roles in terminating glutamatergic transmission (Kanai et al., 2013; Divito and Underhill, 2014). Cellular uptake of glutamate must occur against a steep electrochemical potential gradient (Grewer et al., 2008). Glutamate transporters couple glutamate uptake to the transport of inorganic ions, thereby utilizing the free energy stored as electrochemical potential gradients of these ions to power uphill transport (Grewer et al., 2000). This coupling mechanism is essential for the efficient removal of glutamate from extracellular fluids such as the cerebrospinal fluid, the intestinal lumen and the lumen of renal proximal tubules. It is now generally accepted that three Na + ions and one H+ are co-transported and one K+ is counter-transported (Grewer et al., 2014). Glutamate transporters are thought to play an important role in maintaining the extracellular glutamate concentration at low levels and to protect neurons from the excitotoxic action of glutamate. Any imbalance or deregulation of transporter mechanism leads to increase in extracellular glutamate which follows excitotoxicity. Hypothetical kinetic models for glutamate transporter are shown in Fig. 1. 6. GLT-1 potential target for preventing excitotoxicity Enhanced glutamate uptake is a potential approach to prevent excitotoxicity. Of all types of glutamate transporters, GLT-1/EAAT-1 clears most of the glutamate released in the cortex and hippocampus (Scofield and Kalivas, 2014). Approximately 80% of the glutamate transporters expressed in the hippocampus is GLT-1/EAAT-2 (Mookherjee et al., 2011). Although expressed primarily by astrocytes, GLT-1/EAAT2 is also expressed on neuronal axon terminals. The expression of GLT-1a in axon terminals has potentially important implications for the physiology of excitatory synaptic transmission in regulating synaptic glutamate, maintaining glutamate stores in the presynaptic terminal, interacting with glutamate receptors, contributing a

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Table 1 Historical aspects of glutamate transporter, GLT-1. Findings

References

Glutamate transporters are essential for the removal and termination of action of the excitatory neurotransmitter glutamate from the synapse. Excessive stimulation of glutamate receptors is neurotoxic. Isolation of a cDNA corresponding to the SLC1A2 gene (GLT-1/EAAT2) by screening a human brainstem and cerebellum cDNA library. GLT-1/EAAT2 cloning.

Attwell et al. (1993) and Shashidharan et al. (1994) Choi (1988)

Description of the cloning and characterization of the human GLT-1/EAAT2 promoter, demonstrating elevated expression in astrocytes. Isolation of the cDNA for mouse GLT-1/EAAT2 Isolation of several tissue-specific GLT1 variants from mouse brain and liver and determined that brain and liver GLT1 cDNA clones have different 5-prime ends, corresponding to replacement of 6 N-terminal amino acids in brain GLT1 by 3 N-terminal amino acids in liver GLT1 Cloning of a cDNA corresponding to a GLT1 variant from rat brain The gene is mapped by using FISH technique, which they called glutamate transporter-1 (GLT1) Mapping of the mouse EAAT2 gene to the central region of chromosome 2, where it is located near quantitative trait loci that modulate neuroexcitability and seizure frequency in mouse models of alcohol withdrawal and epilepsy. In mice in which the GLT-1 gene was disrupted in embryonic stem cells by homologous recombination, observed lethal spontaneous seizures and increased susceptibility to acute cortical injury GLT1 was severely decreased in ALS brain tissue, both in the motor cortex (71 to 90% decrease from control) and in the spinal cord. The brains of mice lacking GLAST/GLT-1 developed normally but that GLAST/GLT-1 double-knockout mice died around embryonic days 17 to 18 and exhibited cortical, hippocampal, and olfactory bulb disorganization. Several essential aspects of neuronal development, such as stem cell proliferation, radial migration, neuronal differentiation, and survival of subplate neurons, were impaired. GLT-1 upregulation by ceftriaxone attenuates the hyperalgesia caused by bladder irritation/ inflammation or by neonatal colonic insult GLT-1 cocompartmentalizes with Na+/K+ ATPase, glycolytic enzymes, and mitochondria, providing a mechanism to spatially match energy and buffering capacity to the demands imposed by transport. Increased expression of glutamate transporter GLT-1 in peritumoral tissue associated with prolonged survival and decreases in tumor growth in a rat model Amyloid-β1–42 slows clearance of synaptically released glutamate by mislocalizing astrocytic GLT-1 Glutamate transporters play an important role in redox homeostasis of the brain Ceftriaxone-induced upregulation of glutamate transporter GLT-1 attenuates alcohol withdrawal

Shashidharan et al. (1994) Pines et al. (1992); Arriza et al. (1997) Su et al. (2003)

Kirschner et al. (1994) Utsunomiya-Tate et al. (1997)

a critical role in preventing glutamate mediated excitotoxicity, GLT-1/ EAAT-2 modulates normal synaptic interactions and neural plasticity (Arnth-Jensen et al., 2002; Tsvetkov et al., 2004; Mookherjee et al., 2011). The role of glutamate transporters at glutamatergic synapses is shown in Fig. 2. Dysfunction or reduced expression of GLT-1/EAAT-2 has been documented in both chronic and acute neurodegenerative disorders (Farooqui et al., 2008; Lee et al., 2012a,b). As GLT-1/EAAT2 is the predominant CNS glutamate transporter subtype and pharmacological activation of these would be a powerful tool for increasing the clearance of glutamate in pathological conditions (Scimemi et al., 2013; Di Cairano et al., 2011), it may serve as a novel therapeutic target for the treatment of various neurologic disorders and other diseases. Glutamate release and reuptake balance at the synaptic cleft is shown in Fig. 3. 7. Role of GLT-1 in neurological disorders 7.1. Epilepsy

Schmitt et al. (2002) Takai et al. (1996) Kirschner et al. (1994)

Tanaka et al. (1997)

Rothstein et al. (1995)

Matsugami et al. (2006)

Lin et al. (2011)

Genda et al. (2011)

Sattler et al. (2013)

Scimemi et al. (2013) Robert et al. (2014) Abulseoud et al. (2014)

glutamate regulated anionic conductance to the plasma membrane of the presynaptic bouton, and controlling cross-talk between excitatory synapses. Presence of GLT-1b is also not denied but it is present in less extent (N.H. Chen et al., 2004; W. Chen et al., 2004). Relatively little is known about the mechanisms that regulate GLT-1/EAAT-2 or the other Na+-dependent glutamate transporters, but it suggests that the expression of GLT-1/EAAT-2 is regulated by transcriptional and/or post-transcriptional processes (Robinson, 1998). In addition to playing

Epilepsy is generally a group of disorders characterized by recurrent spontaneous seizures that apparently result from complex processes involving several neurotransmitter systems such as the glutamatergic, cholinergic, and GABAergic systems (de Almeida et al., 2011; Koutroumanidou et al., 2013). According to the World Health Organization (WHO), the estimated prevalence rate for epilepsy is 1–2% of the world population, and despite the fact that there is a considerable number of classic and more modern anticonvulsant drugs available for the pharmacological treatment of epilepsy patients worldwide, seizures remain refractory in more than 20% of the cases (de Almeida et al., 2011). Neuronal excitatory–inhibitory imbalance has been suspected to occur during seizures and to underlie epileptogenesis (Kaila et al., 2013). Excitation is modulated mostly by glutamate (Mehta et al., 2013). After observing epileptic tissue many studies have suggested that malfunctioning of glutamate transporters and astrocytic glutamate converting enzyme, glutamine synthetase (GS), are the main causative factors of hyperexcitation, seizures spread and neurotoxicity (Hammer et al., 2008; Coulter and Eid, 2012; Sun et al., 2013). The glial glutamate–glutamine cycle is a major contributor to synaptic GABA release and regulates inhibitory synaptic strength (Liang et al., 2006). While inhibition of GS did not affect normal glutamatergic transmission, glutamine transport into neurons was required to maintain epileptiform activity (Seifert and Steinhauser, 2013). In the pentylenetetrazol epilepsy model, glutamine-synthetase protein levels were found to be unchanged during immunohistochemistry studies. However, the enzyme underwent stress-induced nitration and partial inhibition in severely affected hippocampal regions (Bidmon et al., 2008; Swamy et al., 2011). Besides, GLT-1/EAAT-2 null mice are prone to exhibit seizures that support GLT-1/EAAT-2 involvement for increased glutamate (Tanaka et al., 1997; Van der Hel et al., 2005). Some studies also reported that an increased GLT-1/EAAT-2 expression can protect mice against SE-induced death, neuropathological changes, and chronic seizure development (Kong et al., 2012; Lopes et al., 2013). In FeCl3induced limbic epilepsy model, GLT-1/EAAT-2 was found to be downregulated on 60th day after initiation of chronic and recurrent seizures in the hippocampus (Ueda et al., 2007). The expression of GLT-1/ EAAT-2 in the hippocampal CA1 region and the motor cortex area was also found to be decreased in chest compression-induced audiogenic epilepsy (Lu et al., 2008). In patients suffering from temporal lobe epilepsy (TLE), increased extracellular glutamate levels in the epileptogenic hippocampus both during and after clinical seizures have been reported. These increased glutamate levels could be the result of malfunctioning and/or downregulation of glutamate transporters. Differences in both mRNA and protein levels of glutamate transporter subtypes were found in specific regions in the TLE hippocampus, with

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Table 2 Classification and distribution of glutamate transporters. Human gene name

Rodent gene name

Cellular expression

Anatomical distribution in nervous system

Anatomical distribution in periphery

EAAT1 EAAT2

GLAST GLT1

Astrocytes Astrocytes

Cerebellum Widespread throughout the CNS

EAAT EAAT4 EAAT5

EAAC1 Rodent EAAT4 Rodent EAAT5

Neurons Neurons Neurons

Widespread throughout the CNS Purkinje cells of the cerebellum Retina

Cochlea, etc. Glandular tissues including mammary gland, hepatocytes and follicular dendritic cells in spleen and lymph nodes. Small intestine, kidney and liver Placenta Liver, kidney, intestine, heart, lung and skeletal muscle

most severe changes found for GLT-1/EAAT2 and EAAT3 levels (Proper et al., 2002). So, on the basis of all the previous reports we can say that enhancing GLT-1/EAAT2 protein expression is a potential therapeutic approach to treat epilepsy. 7.2. Alzheimer's disease (AD) AD is a progressive age-related neurodegenerative disorder. The pathophysiological characteristic of AD is abnormal deposition of fibrillar amyloid β protein, intracellular neurofibrillary tangles, oxidative damage and neuronal death in the brain (Yuan et al., 2014). AD is thought to reflect synaptic dysfunction (Selkoe, 2002). Aberrant glutamate stimulation can cause synaptic dysfunction, which has been

1

+

2

3Na

proposed as one of several mechanisms by which synapses are damaged in AD (Hynd et al., 2004; Mattson, 2003). A number of studies have found that GLT-1/EAAT-2 is significantly reduced or damaged in AD patients (Lauderback et al., 2001; Scott et al., 2011; Mookherjee et al., 2011). On the basis of critical neuroprotective functions carried out by GLT-1/EAAT-2, there raises the possibility that reduced GLT-1/EAAT-2 levels may play a significant role in AD pathogenesis (Woltjer et al., 2010). GLT-1/EAAT-2 contributes to early occurring pathogenic processes associated with AD. Consequently, it is not yet clear whether GLT-1/EAAT-2 dysfunction plays a pathogenic or a bystander role in AD. Partial GLT-1/EAAT-2 loss in an animal model of AD also provokes early-occurring cognitive deficits (Mookherjee et al., 2011), suggesting that GLT-1/EAAT-2 loss is capable of driving cognitive impairment in

Glutamate H (=E/H)

OUT

3Na+

3Na+

+

K

3

E/H

IN Na+ Leak

Relocation Re

Translocation

OUT

+

3Na+

K

3Na+

E/H

IN 6

+

K

+

5

F/H

4

Fig. 1. Hypothetical kinetic models for glutamate transporter. Under normal conditions, glutamate transport involves loading the empty glutamate carrier with glutamate/H+ and 3 Na+, followed by translocation of the fully loaded carrier across the plasma membrane (charge translocation step) and release of the substrates at the intracellular face. Thereafter, K+ binds to the carrier inside and promotes the relocation of the empty carrier. For net uptake of glutamate, glutamate transporters have to complete this cycle. If it is not completed because the empty carrier cannot enter the relocation step, the empty carrier binds Na+ and glutamate again at the inside of the cells and translocates back in the reverse direction. In this case, the transporter behaves like an exchanger.

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Neuron

Glia

Glia

(Presynapc terminal) ATP

ADP Glu

-

Adrenoceptor Glu

-

Glu

-

Gln

-K

Gln

+

Glu

Gln

Glu

Gln

-

GLT1/EAAT2 +

H

GLAST/EAAT1 3Na+

ATP -

Glu GLT1/EAAT2

K

+

2+

Ca

+

Na

3Na+

NMDA

AMPA

2+

EPSP

(Excitatory post synapc potenal)

Ca

+

H

Glu +

K

GLAST EAAT1

+

H +

3Na

EAAT3

Neuron (Post-synapc terminal) P2XR

Fig. 2. Role of glutamate transporters at glutamatergic synapses. The excitatory neurotransmitter L-glutamate is stored in synaptic vesicles at presynaptic terminals. Glutamate is transported into these vesicles by the vesicular glutamate transporters. Glutamate is released into the synaptic cleft to act on glutamate receptors. The AMPA receptors mediate fast excitatory postsynaptic potentials (EPSP), whereas the NMDA receptors possess a cation channel that is permeable to Ca2+. High affinity glutamate transporters play essential roles in removing released glutamate from the synaptic cleft. These transporters are also crucial for maintaining the extracellular glutamate concentration of the cerebrospinal fluid (CF) below neurotoxic levels.

the context of Aβ-related neuropathology (Schallier et al., 2011). Whether Aβ-related pathogenic processes have a functional impact on the rate at which astrocytes remove endogenous, synaptically released

glutamate is unknown (Scimemi et al., 2013). It was previously noted that Aβ1–42 induces rapid GLT-1/EAAT-2 mislocalization and internalization in astrocytes, which leads to a marked reduction in the

Fig. 3. Glutamate release and reuptake balance at the synaptic cleft.

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rate at which astrocytes clear up synaptically released glutamate from the extracellular space (Scimemi et al., 2013). Thus, many findings and reports indicated that astrocytic GLT-1/EAAT-2 dysfunction may play an important role in the pathogenesis of AD and suggested the possible mechanisms by which several current treatment strategies could protect against the disease (Schallier et al., 2011).

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not clear yet, whether the loss of the transporter is a consequence of the neurodegeneration induced by mutant SOD1 or whether the presence of mutant SOD1 protein in astrocytes is sufficient to affect the expression of GLT-1/EAAT-2. Downregulation of GLT-1/EAAT-2 by increasing the internalization and degradation of the surface transporter is found to be the major cause which leads to ALS. So, it may serve as an important therapeutic target for the treatment of ALS (Vanoni et al., 2004).

7.3. Stroke Stroke is the third leading cause of death in industrialized countries and the most frequent cause of permanent disability in adults worldwide (Varsou et al., 2014; Lo et al., 2003; Donnan et al., 2008). Stroke is primarily either hemorrhagic or ischemic, and almost 80% of the stroke is ischemic (Banerjee et al., 2011). Aberrant function of glutamate transport plays an essential role in the excitotoxic neurodegeneration that occurs in the model of cerebral ischemia (Takatsuru et al., 2014; Chao et al., 2010). There is tenfold higher concentration of glutamate within the cells. The energy and ion gradient necessary to maintain this state fail under ischemic condition (Nishizawa, 2001). Release of glutamate occurs within minutes of ischemic onset. Glutamate transporter down-regulation may disrupt the normal clearance of the synaptically-released glutamate and may contribute to the ischemic neuronal death (Rao et al., 2001). The inhibition of glutamate-induced excitotoxicity has been a therapeutic target for the treatment of stroke (Harvey et al., 2011). Many studies had extend the pharmacological manipulation of GLT-1/EAAT-2 levels (Rothstein et al., 2005; Chu et al., 2007) to genetic manipulation of GLT-1/EAAT-2 as a gene therapy approach to reduce ischemia-induced excitotoxicity. Increased expression of GLT-1/EAAT-2 reduces the damage caused by brain ischemia. Overexpression of GLT-1/EAAT-2 in the ischemic cortex reduces the size of lesion and improves the behavioral recovery (Harvey et al., 2011). The modulation of GLT-1/EAAT-2 has been shown to exert neuroprotection in various models of ischemic injury and motor neuron degeneration (Mogoanta et al., 2014). An attempt to explore the neuroprotective potential in cerebral ischemia/reperfusion injury was also made by using ceftriaxone, a GLT-1/EAAT-2 modulator which provides overwhelming evidence that modulation of GLT-1/ EAAT-2 protein expression and activity confers neuroprotection in cerebral ischemia/reperfusion injury (Verma et al., 2010). Therefore, these may serve as an important target for stroke also. In contrary, Rossi et al. (2000) found that transporter-mediated glutamate homeostasis fails dramatically in ischemia but instead of removing extracellular glutamate to protect neurons, transporters release glutamate triggering neuronal death. In this case, to use GLT-1 modulators may potentiate neuronal death and complexity of the disease. 7.4. Movement disorders 7.4.1. Amyotrophic lateral sclerosis (ALS) It is an adult-onset, chronic neuromuscular disorder characterized by the selective degeneration of cortical and spinal/bulbar motor neurons. Approximately 10% of the cases are hereditary (familial ALS), 15–20% of which are associated with mutations in the gene Cu2 +– Zn2+ superoxide dismutase (SOD1) (Vanoni et al., 2004). Among the various mechanisms proposed as initiator or potentiator of neuronal damage in ALS, glutamate excitotoxicity and oxidative damage seem to be correlated and mutually reinforcing (Julien, 2001; Cleveland and Rothstein, 2001). A number of studies have documented the loss of GLT-1/EAAT-2 protein in the motor cortex and spinal cord of patients with sporadic or familial ALS and abnormal glutamate metabolism (Rothstein et al., 1995; Bristol and Rothstein, 1996). The specific loss of the astroglial GLT-1/EAAT-2 has also been documented in some animal models of ALS expressing SOD1 mutants which is highly relevant to the pathogenesis of ALS (Bruijn et al., 1997; Bendotti et al., 2001). It is

7.4.2. Parkinson's disease (PD) Human idiopathic Parkinson's disease (PD) is a progressive neurodegenerative movement disorder that is primarily characterized by degeneration of the dopaminergic neurons at the nigrostriatal pathway (Deumens et al., 2002). The excitatory amino acid glutamate has been suggested to play a central role in the pathophysiology of Parkinson's disease. In PD, the degeneration of dopaminergic neurons is believed to lead to an overactivation of the subthalamic nucleus (Rodriguez et al., 1998). This increases the firing rate of the glutamatergic excitatory projections to the substantia nigra (Bamford et al., 2004; Ambrosi et al., 2014). This sustained exposure to glutamate could accelerate the degeneration of dopaminergic neurons by intense stimulation of Nmethyl-D-aspartate (NMDA) and 2-amino-3-hydroxy-5-methyl-4isoxazolepropionate (AMPA) receptors (Oster et al., 2014). Even though an exact conclusion for the role of glutamate transporters in the pathogenesis of the disease is not found, some studies do clearly demonstrate a link between disturbed glutamatergic neurotransmission and glutamate transporter functioning in the striatum of an animal model for PD (Massie et al., 2010). Dopamine neurons express the neuronal transporter EAAC1 (EAAT3) at high levels (Plaitakis and Shashidharan, 2000), and during pathological conditions which lead to excessive neuronal depolarization, it can affect or even reverse EAAT function by altering the transport driving force (Danbolt, 2001; Assous et al., 2014). In a few studies, a time-dependent bilateral effect of unilateral 6-hydroxydopamine lesioning on the expression as well as activity of GLT-1 was found in animal models of PD (Carbone et al., 2012). In 6-hydroxydopamine (6-OHDA), L -DOPA induced PD model it was also reported that increasing glutamate uptake by GLT-1 may prolong the time period before locomotor impairment occurs (Chotibut et al., 2013; Kelsey and Neville, 2014). Based on previous studies and data, GLT-1/EAAT-2 may be a potential target for PD. 7.4.3. Huntington's disease (HD) HD is a progressive autosomal dominant neurodegenerative disorder that is caused by a CAG repeat expansion in the HD gene resulting in an expansion of polyglutamines at the N-terminus of the huntingtin protein and accumulation of the mutant protein as cytoplasmic and nuclear aggregate inclusions (Bates, 2003; Walker, 2007). HD is pathologically characterized by degeneration in neostriatal (caudate and putamen) and cerebral cortex that is believed to be the underlying contributor of motor impairment, cognitive decline and psychiatric symptoms that worsen as the disease progresses. Oxidative stress due to mitochondrial dysfunction has been implicated in human patients and HD animal models (Browne and Beal, 2006). Defects in mitochondrial complexes II, III and IV were observed in striatum of post-mortem HD brain (Aidt et al., 2013; Damiano et al., 2010). The distribution of glia-bound GLT-1 mRNAs was investigated in postmortem brains of HD patients. GLT-1mRNA was found to be decreased in correlation to disease severity (Arzberger et al., 1997; Faideau et al., 2010). Excitotoxicity is thought to be important in the pathogenesis of HD (Miller et al., 2008; Shin et al., 2005). GLT-1/EAAT-2 is the most abundant glutamate transporter, and accounts for most of the glutamate transport in the brain. It was found that increase in the functional expression of GLT-1/EAAT-2, can improve the behavioral phenotype of the mouse model of HD (Miller et al., 2012; Estrada-Sanchez et al.,

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2009). To test the hypothesis that GLT-1/EAAT-2 expression critically affects the HD disease process, a novel mouse model R6/2 transgenic model that is heterozygous for the null allele of GLT-1/EAAT-2 and carries the double mutation was generated and it was found that the protein expression of total GLT-1/EAAT-2, as well as two of its isoforms, is decreased within the cortex and striatum of 12-week-old R6/2 mice (Petr et al., 2013). These study results suggested that changes in GLT1/EAAT-2 expression or function can potentiate or ameliorate the progression of HD and it can serve as an important target for HD treatment (Petr et al., 2013). Growing evidence also indicates that ascorbate (AA), an antioxidant vitamin, is released into striatal extracellular fluid when glutamate is cleared after its release from cortical afferents. Both AA release and glutamate uptake are impaired in the striatum of transgenic mouse models of HD owing to a downregulation of GLT-1/EAAT-2 (Rebec, 2013). 7.5. Psychosis Schizophrenia is a debilitating psychiatric illness that affects approximately 1% of the world's population. The main symptoms associated with schizophrenia are grouped into three major symptom clusters that include positive symptoms, negative symptoms, and cognitive disturbances (Shan et al., 2012). Dopaminergic agents have been the mainstay of antipsychotic drug therapy for decades (Miyamoto et al., 2005). Current drug therapies for schizophrenia include typical (e.g., haloperidol and chlorpromazine) and atypical (e.g., olanzapine and clozapine) antipsychotics, all of which act as antagonists at D2 dopamine receptors and other monoamine receptor subtypes. Typical and atypical antipsychotics are at least partially effective in reducing the positive symptoms in most patients. However, some patients are refractory and these drugs are not effective in the treatment of the negative and cognitive symptoms (Lieberman et al., 2005). In addition, available antipsychotic medications induce metabolic syndrome, Parkinsonian-like motor effects, and other adverse effects that are tied to their mechanism of action (Lieberman et al., 2003; Pramyothin and Khaodhiar, 2010). Recent attention has focused on the role of glutamate in schizophrenia (Poels et al., 2014) and many components of the glutamate synapse represent evolving molecular targets. Among the proteins regulating glutamate availability in the synapse, EAATs, might offer an approach to manipulate the glutamate synapse as a strategy for antipsychotic drug development but the efforts targeting the glutamate transport system have been limited (Dunlop and Marquis, 2006). Partial deletion of the EAAT-1 gene has also been found in schizophrenia patients (Walsh et al., 2008), while expression of a high-risk for schizophrenia allele of the GRM3 metabotropic glutamate receptor is associated with decreased EAAT-2 mRNA expression in human prefrontal cortex (Egan et al., 2004; Walsh et al., 2008). Taken together, these findings suggest that abnormal expression of GLAST/EAAT-1 and GLT-1/EAAT-2 may contribute to the pathophysiology of schizophrenia. A rigorous evaluation of the utility of GLT transporters as potential antipsychotic drugs awaits the development of compounds with improved selectivity and pharmacological properties and their evaluation in a broad spectrum of animal models predictive of antipsychotic efficacy. 8. GLT-1 modulators (shown in Table 3) 8.1. Ceftriaxone Although beta-lactams have been historically used as antimicrobials, a notable ancillary effect in the host was identified by Rothstein et al. (2005). The beta-lactams antibiotics, such as ceftriaxone, are shown to enhance the ex vivo expression of a neuroprotective protein GLT-1/ EAAT-2 in a concentration dependent manner (Karaman et al., 2013). GLT-1/EAAT-2 terminates the potentially neurotoxic effects of the

neurotransmitter glutamate by removing it from the synaptic cleft. GLT-1/EAAT-2 plays an integral role in both normal and abnormal neurotransmission (Guo et al., 2003). Beta-lactam-mediated activation of GLT-1/EAAT-2 expression is proposed to involve nuclear factor-kB (NF-kB), whereby β-lactams induce the nuclear activation of this transcription factor (Feng et al., 2014). Activated NF-kB then binds to the GLT-1/EAAT-2 promoter region and up-regulates the transcription of this gene (Lee et al., 2008), thereby decreasing glutamate concentration in the synaptic cleft and alleviating the potentially neurotoxic effects of excessive glutamate (Salles et al., 2014). In some studies it was also found that ceftriaxone treatment can attenuate neuronal injury and improve spatial learning and memory after chronic cerebral hypoperfusion and that glutamate excitotoxicity may play an important role in the pathophysiology of chronic cerebral hypoperfusion (Koomhin et al., 2012). This ceftriaxone induced GLT-1/EAAT-2 up-regulation blocks the metabotropic glutamate receptor (mGluR) dependent long-term depression (LTD) at the mossy fiber (MF)–CA3 hippocampal synapse. It also has negative effects on long-term potentiation (LTP). GTs provide an essential regulatory role in hippocampal learning and memory (Matos-Ocasio et al., 2014).

8.2. Tamoxifen (TX) A selective estrogen receptor modulator may act as an agonist in brain tissue. TX enhanced the expression and function of GLT-1/EAAT2 in rat astrocytes (Lee et al., 2012a,b). TX phosphorylated the cAMP response element-binding protein (CREB) and recruited CREB to the GLT-1/EAAT-2 promoter consensus site. The effect of TX on astrocytic GLT-1/EAAT-2 was attenuated by the inhibition of PKA, the upstream activator of the CREB pathway (Karki et al., 2013). In addition, the effect of TX on GLT-1/EAAT-2 promoter activity was abolished by the inhibition of the NF-κB pathway (Ghosh et al., 2011). Furthermore, TX recruited the NF-κB subunits p65 and p50 to the NF-κB binding domain of the GLT-1/EAAT-2 promoter. Taken together, it was established that TX regulates GLT-1/EAAT-2 via the CREB and NF-κB pathways (Karki et al., 2013).

8.3. 17β-estradiol (E2) 17β-Estradiol (E2) is one of the most active estrogen hormones possessing neuroprotective effects in both in vivo and in vitro models, and it has been shown to enhance astrocytic glutamate transporter function (Liang et al., 2002). E2 has been shown to increase expression of both GLAST/EAAT-1 and GLT-1/EAAT-2 mRNA and protein and glutamate uptake in astrocytes (Lee et al., 2013). Growth factors such as transforming growth factor-α (TGF-α) appear to mediate E2-induced enhancement of these transporters. Therefore, there might be astrocytemediated E2 neuroprotection, with a focus on glutamate transporters (Karki et al., 2014).

8.4. Recombinant EGF The glutamate transporter gene, GLT-1/EAAT-2, is induced by epidermal growth factor (EGF) and downregulated by tumor necrosis factor alpha (TNFα) (Lee et al., 2013). EGF-mediated activation of GLT-1/EAAT-2 expression requires NF-kappaB (Sitcheran et al., 2005).

8.5. Group III metabotropic receptor antagonist It was reported that Group III metabotropic receptor antagonist significantly restored the GLT-1/EAAT-2 levels in brain and has a potential application in neurodegenerative disorders (Chen et al., 2005; Michele and Fiorenzo, 2010).

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Table 3 Various drugs and their mechanisms by which they up-regulate and down-regulate the GLT-1 expression. Drugs

Mechanism of action

References Verma et al. (2010); Abulseoud et al. (2014)

Recombinant EGF Group III metabotropic receptor antagonists (S-α-MCPA, L-AP3) Minocycline

NF-κB activation and P65 nuclear translation through proteasomal degradation of IkBα Increases TGF-α expression Increases mRNA and protein levels of transporters and upregulation of growth factors (TGF-α, TGF-1) Phosphatidylinositol and NF-κB activation Restores the levels of GLT-1 by upregulating its mRNA and protein levels in hippocampus and striatum GLT-1 upregulation

Down-regulators Clozapine

Synaptophysin upregulation

Chronic morphine Dihydrokinic acid Endothelins

Changes GLT mRNA expression GLT-1 blocker Loss of GLT-1 expression

Up-regulators Ceftriaxone Tamoxifen 17 β-Estradiol

8.6. Minocycline The prevention of downregulation of glial GTs by minocycline in neuropathic rats most likely results from the inhibition of glial activation and the subsequent release of bioactive substances from activated glial cells by minocycline (Nie et al., 2010). The inhibition of glial cell activation by minocycline and the subsequent suppression of both the production of proinflammatory cytokines and the activation of NF-κB and nitric oxide synthase result in the preservation of glial GT expression (Ye and Sontheimer, 1996). Minocycline preserves the normalized activation of glutamate receptors and synaptic transmission by maintaining both the expression (number) of glial GTs and the availability of glial GTs to presynaptically released glutamate (Nie et al., 2010).

Lee et al. (2012a,b) Pawlak et al. (2005); Lee et al. (2013) Zelenaia et al. (2000) Chen et al. (2005) Nie et al. (2010)

Vallejo-Illarramendi et al. (2005); Bragina et al. (2006) Lim et al. (2005) Karaman et al. (2013) Rozyczka et al. (2004)

clinical trials. It is our great hope that the GLT-1/EAAT-2 treatment strategy will be successful and greatly benefit patients suffering from severe neurodegenerative diseases without negative side effects. Mechanism of action of ceftriaxone is shown in Fig. 4. 10. Conclusion The concept of the role of GLT-1/EAAT-2 in preventing excitotoxicity as a main target to treat neurotoxicity has made great progress in determining its role in the pathogenesis and treatment of neurodegenerative diseases. It is now accepted that GLT-1/EAAT-2 transporters are the major targets to combat neurotoxicity and are the novel potential targets for the treatment of neurological diseases. Acknowledgment

9. Future prospective L-Glutamate

mediated excitotoxicity is involved in a wide range of acute and chronic neurodegenerative diseases such as epilepsy, stroke, AD, PD, and ALS. Presently there is no any safe and effective drug for the prevention of excitotoxicity. Many glutaminergic synapse acting drugs are available to treat neurological disorders like glutamate receptor antagonists which have been tried to treat stroke, topiramate (glutamate receptor antagonist), riluzole preventing glutamate release, is (approved for ALS treatment), but it has not been a very successful strategy in humans because it affects normal brain function and produces negative side effects. Loss of GLT-1/EAAT-2 protein and function is approximately found in all chronic neurodegenerative diseases and on the basis of previous reports it may be the main cause of excitotoxicity in these diseases. Therefore, restoring GLT-1/EAAT-2 protein levels and function may provide therapeutic benefits. It is hypothetically proved that GLT-1/EAAT-2 can be up-regulated by transcriptional or translational activation. Reported transcriptional activators of GLT-1/EAAT-2 i.e. ceftriaxone which is a β lactam antibiotic have been tested in many disease models and are capable of providing neuronal protection. LDN212320 is also one of the newly identified GLT-1/EAAT-2 modulators, and research is still going to develop or identify more GLT-1/ EAAT-2 modulators with improved pharmacological and bioactivity properties. Ceftriaxone is currently in human clinical trials for ALS (Zhao et al., 2014). Transition from preclinical mouse studies to human clinical trials is very difficult; successful preclinical studies often fail in subsequent

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Fig. 4. Mechanism of action of ceftriaxone.

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