The role of glutamate transporter EAAT2 in the treatment of neurodegenerative diseases.
Glutamate, also known as glutamic acid is the major amino acid in the human body and it is most abundantly present in the brain and muscles. It is the principal excitatory neurotransmitter in the brain (1). Glutamate is the principal mediator of sensory information, motor coordination, emotions, and cognition, including memory formation and memory retrieval (2). Glutamate is zwitterionic and cannot diffuse across the membranes, it must be synthesized and metabolized within CNS. Therefore, uptake mechanisms come into action via glutamate transporters (3,4). Glutamate transporters, also known as excitatory amino acid transporters (EAATs) are sodium and potassium-dependent transporters and are distributed throughout the brain (5). Abnormal distribution of glutamate or failure in proper glutamate clearance leads to excitotoxicity. The glial glutamate transporter EAAT2 plays a significant role in glutamate clearance and decrease glutamate clearance due to reduced expression of EAAT2 has been attributed to many neurodegenerative diseases such as stroke, Parkinson’s disease, epilepsy, amyotrophic lateral sclerosis, Alzheimer’s disease, major depressive disorder, and addiction (6,7). Therefore, EAAT2 can be a great target that can be stimulated to increase glutamate uptake, thereby, preventing its overaccumulation in the synaptic cleft.
Glutamate is the most abundant excitatory neurotransmitter in the central nervous system as well as a key metabolite linking carbon and nitrogen metabolism (8). The synthesis of the amino acid is carried out via a process called transamination. This involves the transfer of a α-amino group from an amino acid to the α-keto position of a α-keto acid. The enzyme that catalyzes this reaction is called an aminotransferase and this enzyme is very critical for the production of non-essential amino acids, such as aspartate, glutamate, and alanine (4). Glutamate is zwitterionic and cannot diffuse across the membranes, it must be synthesized and metabolized within CNS (3). Glutamate is the principal mediator of sensory information, motor coordination, emotions, and cognition, including memory formation and memory retrieval (2). Glutamate is stored in vesicles in presynaptic terminals. It is released from pre-synaptic terminals by increased intracellular calcium concentration and diffuses across the synaptic cleft where it binds glutamate receptors on the postsynaptic terminals and gives rise to excitatory postsynaptic potential (EPSP) (9,10). There are two types of glutamate receptors depending on the mechanism by which they generate postsynaptic current and they are ionotropic receptors (iGluRs) and metabotropic receptors (mGluRs) (10). iGluRs are a group of receptors that are related to their amino acid sequences and belong to the huge superfamily of ion channels containing a P-loop as the ion-pore-forming segment. The iGluR family consists of four subgroups: AMPA, NMDA, kainate, and orphan receptors. Each subgroup has a unique function and the AMPA receptors are the ones that are widely expressed all over the brain (11). The metabotropic glutamate receptors (mGluRs) are family C G-protein-coupled receptors that participate in the modulation of synaptic transmission and neuronal excitability throughout the central nervous system (12). The glutamate is cleared from the extracellular fluid via uptake mechanisms by a special protein family known as excitatory amino acid transporters, EAATs (9,10). After glutamate uptake into glial cells by the EAATs, it is converted to glutamine and then transported back into the presynaptic neuron where it is converted back into glutamate, and taken up into synaptic vesicles via vesicular glutamate transporters, VGLUTs (19,20). There are five different subtypes of EAAT proteins and have different nomenclature, distribution, expression patterns and uptake kinetics (9). They are EAAT1, EAAT2, EAAT3, EAAT4 and EAAT5. Among the five subtypes, the glial carriers, EAAT1 and EAAT2 have the significant impact on clearance of glutamate released during neurotransmission and are expressed in astroglial cells (13,14). The EAAT3-4 subtypes are expressed in axon terminals, cell bodies, and dendrites (15,16). EAAT5 is only found in the retina where it is principally localized to photoreceptors and bipolar neurons (17). The glutamate transporter EAAT2 is responsible for 90% of total glutamate uptake and its dysfunction can lead to development of various neurodegenerative diseases including stroke, Parkinson’s disease, epilepsy, amyotrophic lateral sclerosis, Alzheimer’s disease, major depressive disorder, and addiction (6,7,18).
EAAT2: Expression and Regulation.
Excitatory amino acid transporter 2 (EAAT2) is also known as solute carrier family 1 member 2 (SLC1A2) and glutamate transporter 1 (GLT-1) and this protein in humans is encoded by the SLC1A2 gene (21,22). The glutamate transporter subtype EAAT2/GLT-1 is expressed throughout the brain primarily in astroglial cells and contribute to 90% of the total glutamate uptake. (13,14,18). The EAAT2 gene is composed of 11 exons which form multiple splice variants (9). Multiple GLT1/EAAT2 mRNA transcripts have been identified in vitro and in vivo, and these transcripts give rise to differential GLT1/EAAT2 splice variants (23). The gene for EAAT2 has two carrier isoforms (EAAT2a and EAAT2b) having distinct C termini as a consequence of alternative RNA splicing (24). The first isoform EAAT2a is expressed by astrocytes throughout the brain as well as a small subset of neurons, whereas the second isoform, EAAT2b, is found in astrocytes and other glial cells (24). The PDZ-binding domain of EAAT2b, a disc large homolog 1 protein found in a variety of neuronal and glial cell type, is involved in the interaction between EAAT2b and the PDZ domain protein, PICK1, thereby affecting their trafficking and synaptic plasticity (24,25). Increase in transporter function is contributed to the neuronal release of glutamate and also the distribution of EAAT2 but is independent of transporter synthesis and glutamate receptor activation (25).
EAAT2 expression is dynamically regulated at the level of transcription, translation, trafficking, transport, and degradation (9). Regulators of EAAT2 transport, both positive and negative, alter EAAT2 transcription, promoter activity, mRNA, and protein (26). The EAAT2 promoter consists of various transcription factor-binding sequences and they are NF-κB, Sp1, N-myc, CREB, EGR, and NFAT (26,27). NF-κB signaling has also been linked to amitriptyline-dependent upregulation of EAAT2 in vivo (27). TNF-alpha, a positive regulator of NF-κB dependent gene expression, downregulates the glutamate transporter EAAT2 and epidermal growth factor (EGF) stimulates EAAT2 (28). TNF-alpha-mediated repression through a distinct pathway, recruiting N-Myc binding sites and EGF-mediated activation of EAAT2 expression require NF-kappaB. The ability of NF-κB to regulate EAAT2 expression has important implications for the regulation of glutamate homeostasis in the CNS (28). EAAT2 protein is highly regulated at the translational level and translation is attributed to corticosterone in primary astrocyte cell lines, primary cortical neuron–astrocyte mixed cultures, and mice (29).
EAAT2: Physiological Roles
EAAT proteins play a significant role in maintaining low extracellular L-glutamate levels and prevent excess glutamate accumulation outside the cells. Overaccumulation of glutamate causes calcium ions to enter cells via NMDA receptor channels, leading to the production of reactive and excitotoxic oxygen/nitrogen species, which induce oxidative stress leading to neuronal death, and is called excitotoxicity (10,18). EAAT2 being responsible for the major amount of glutamate uptake is primarily involved in tightly regulating glutamate concentration in the synaptic cleft and thus dysfunction of EAAT2 transporter has a greater impact in the generation of neurodegenerative disease (18). It has been possible to emphasize the function of EAAT2 transporters due to the availability of selective pharmacological inhibitors, such as dihydrokainate (30). Rothstein and his colleagues showed that impaired glutamate transporter expression contributed to neurodegeneration in the normal animal using anti-sense knock-down (31). Also in a study, Tanka and his colleagues found that EAAT2 knock out mice exhibited significantly reduced transport activity, seizures, and increased sensitivity to neurotoxicity and this suggest that impaired glutamate transport can lead neurodegeneration (32). Dysfunctional or reduced expression of EAAT2 protein is mostly observed in chronic neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), Parkinson’s disease (PD), epilepsy, cerebral ischemia and Alzheimer’s disease (AD) and could be the possible factor behind excitotoxicity in these diseases (30). EAAT2 expression is dynamically regulated at the transcription and post-transcriptional level and EAAT2 function can be pharmacologically modulated with moderate specificity which indicates EAAT2 could be a great target for combating neurodegenerative disease (9).
Neurodegeneration is a process which leads to irreversible neuronal damage and death and a common final pathway present in aging and neurodegenerative diseases (33). Neurodegenerative diseases are characterized by a significant loss of neurons that often leads to death (34). A wide range of neurodegenerative disorder affects the brain or spinal cord of humans. The degeneration of nervous tissue can result from both, acute injury and from chronic disease. Acute neuropathology in adults, children, and infants is caused by head or spinal cord trauma, infection, toxicity, liver failure, and cerebral ischemia resulting from stroke, cardiac arrest, or asphyxiation (35). Chronic, progressive neuropathology is observed in adult disorders such as Alzheimer’s disease, amyotrophic lateral sclerosis (Lou Gehrig’s disease), Huntington’s disease, multiple sclerosis, and Parkinson’s disease and in infants and children with spinal muscular atrophy (35). Many neurodegenerative diseases are genetically inherited. The recent advances in genetics have enabled to identify many causative genes for neurodegenerative diseases and this information can be very useful is designing knock-out animal models for further study and the better understanding of the disease (36).
Alzheimer’s is the most common type of dementia, accounting for 60-80% of dementia cases. It leads to problems with memory, thinking, behavior and the symptoms usually develop and gradually become severe in a way to interfere with daily activities (37). Worldwide, nearly 44 million people have Alzheimer’s or related dementia. Alzheimer’s and other dementias are the number one cause of disabilities in later life (38). Alzheimer’s disease (AD) is the sixth-leading cause of death in the United States and an estimated 5.5 million Americans of all ages have Alzheimer’s disease (39). Glutamate toxicity is found to have a role in neurodegeneration in Alzheimer’s disease. The AD is characterized by the presence of extracellular amyloid plaques, consisting mostly of the β-amyloid peptide (Aβ), and intraneuronal aggregates of the protein tau (40). The role of plaques and tangles in Alzheimer’s disease pathophysiology is still not very clear. However, it is believed to play a critical role in blocking communication among nerve cells and disrupting processes that cells need to survive (37). Reduced glutamate uptake function may result in increased extracellular glutamate levels which, in turn, potentially increase amyloid-b production over time (9). Shaomin Li and his colleagues reported that Aβ oligomers from several sources (synthetic, cell culture, human brain extracts) facilitate electrically-evoked long-term depression (LTD) in the CA1 region and the Aβ-enhanced LTD was due mGluR or NMDAR activity, depending on the induction protocol (41). Further, both forms of LTD were prevented by an extracellular glutamate scavenger system (41).
The principle glutamate transporter EAAT2 has a crucial role in cognitive functions and EAAT2 is found to be diminished in AD patients. This may be because of disruption at the post-transcriptional level as EAAT2 mRNA is not diminished in AD patients (42). Glutamate transporters are also decreased in animal models of the AD. Masliah E et al., in a study with transgenic mice expressing a mutant form of human amyloid precursor protein observed a significant reduction in aspartate binding, GLAST protein, and GLT-1/EAAT2 protein compared to control group. This decrease in glutamate transporter function was found to be associated with decreased protein expression of glial-specific glutamate transporters, EAAT1 and 2, but did not affect mRNA levels (43). Therefore, stimulating EAAT2 expression through translational mechanisms could be a potential approach to drug discovery for the AD.
Parkinson disease (PD) is the world’s second-leading devastating, relentlessly progressive neurodegenerative disorder characterized by motor impairment (such as bradykinesia and resting tremor) and dementia (44,47). Parkinson’s disease (PD) affects 1-2 per 1000 of the population at any time and its prevalence is increasing with age thereby affecting 1% of the population above 60 years (48). The etiology of PD is mostly unknown, but it likely involves both genetic and environmental factors (45). PD is characterized by loss of dopaminergic cells and occurrence of Lewy bodies in the substantia nigra and specific brain stem areas (46).
The dopamine and glutamate in the basal ganglia play a significant role in regulating critical aspects of motor and cognitive behavior. The degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) in Parkinson’s disease (PD) leads to excessive activity of glutamatergic neurons of the subthalamic nucleus (STN) and is believed to result in the motor symptoms of PD (49). Metabotropic glutamate (mGlu) receptors are responsible for glutamatergic dysfunction in Parkinson’s disease (PD) (49). Treatment option consists of drugs to increase dopamine and therefore Levodopa and carbidopa are commonly used drugs for Parkinson’s disease. L-DOPA treatment leads to dyskinesias further increases basal levels of glutamate function in basal ganglia (49). This overexpression of GLT1/EAAT2 induced by L-DOPA could represent a compensatory mechanism to limit glutamate overactivity due to high levels of extracellular glutamate (30). Thus, blocking excessive glutamate transmission could alleviate symptoms and delay progression of the disease. Both preclinical and clinical studies demonstrate that glutamate receptors are a therapeutic target for PD as it could alleviate the motor symptoms of PD and also reduce the onset of levodopa-induced dyskinetic motor behavior (50). EAAT2 expression has also found to be reduced in animal models of PD, 6-hydroxydopaminelesioned PD model and the acute 1-methyl-4-phenyl1,2,3,6tetrahydropyridine treated mouse model (51,52). Therefore, GLT-1/EAAT2 could be a promising target for Parkinson’s disease.
Amyotrophic lateral sclerosis
ALS, or amyotrophic lateral sclerosis, the rare group of progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. Based on U.S. population studies, a little over 6,000 people in the U.S. are diagnosed with ALS each year. (That’s 15 new cases a day.) It is estimated there are more than 20,000 Americans have the disease at any given time. According to the ALS CARE Database, 60% of the people with ALS in the Database are men and 93% of patients in the Database are Caucasian (53). The disorder is named after its underlying pathophysiology, with “amyotrophy” referring to the atrophy of muscle fibers, which are denervated as their corresponding anterior horn cells degenerate (54). Some of the cellular processes that occur after disease onset, have been identified and that includes mitochondrial dysfunction, protein aggregation, generation of free radicals, excitotoxicity, inflammation, and apoptosis, but for most patients, the underlying cause is unknown (55).
Glutamate transport is found to be diminished in the motor cortex and the spinal cord of tissue from amyotrophic lateral sclerosis (ALS) patients. This defect appears to be due to a selective loss of the astroglial specific glutamate transporter protein EAAT2 (56). Overall, 30–95 % loss of the EAAT2 protein has been observed in the motor cortex and spinal cord in approximately 60–70 % of ALS patients (57). The loss of EAAT2 protein is also observed in a transgenic animal model of mutant SOD1-mediated familial ALS and the loss of GLT-1 protein selectively occurs in the areas affected by neurodegeneration and reactive astrocytosis and it is not associated with increases of glutamate levels in CSF (58). These findings suggest that enhanced EAAT2 expression could be useful in treating ALS. However, ceftriaxone, known to enhance EAAT2 expression did not show great results in clinical trials (9). This may be due to other complex factors associated with the disease which is somehow interfering the upregulation of EAAT2 in presence of ceftriaxone. Therefore, the EAAT2 level, pre, and post-treatment need to be studied and this can provide an important information about the efficacy profile.
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease. It is characterized by mental decline, psychiatric disorder and motor dysfunction predominantly chorea, an involuntary movement disorder. HD is caused by a highly polymorphic CAG trinucleotide repeat expansion in the exon-1 of the gene encoding for huntingtin protein (59). This mutation leads to degeneration of the striatum and deep cortical layers, and eventually, the hippocampus and hypothalamus (30). HD is characterized by protein aggregates that accumulate within cells like that in various forms of spinocerebellar ataxia, as well as in other neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) (59). HD is reported to be prevalent one in every 10,000 persons and nearly 30,000 in the United States have Huntington’s disease. Juvenile Huntington’s occurs in approximately 16 percent of all cases (60).
Mutant huntingtin affects the glutamatergic system. Mutant huntingtin with the polyQ expansion is associated with reduced binding to post-synaptic density protein 95 (PSD-95), which allows PSD-95 binding with NMDA receptors at the cell surface which stabilizes and increases receptor activation. This shows overactive glutamate receptors has a critical role in the pathogenesis of HD (61). The abnormal neuronal function is the major factor contributing to HD behavioral phenotype, and dysregulation of glutamate is believed to play a critical role (62). Decreased glutamate uptake is observed in the prefrontal cortex of HD postmortem tissue, and this effect is seen in the early stages of HD (63). Also, palmitoylation is critical for normal GLT-1/EAAT2 function and impaired GLT-1 palmitoylation in a mouse model is found early in the HD pathogenesis which may lead to excitotoxicity, and ultimately, neuronal cell death in HD (64).
Epilepsy is a chronic disorder characterized by recurrent seizures which may be a result of brain injury, but the exact cause is still not clear. It is currently estimated that around 2.2 million people in the United States suffer from Epilepsy (65). Epileptic seizures can be categorized into two different type and they are generalized seizures and partial seizures. Generalized seizures exhibit a widespread electrical discharge in both brain hemispheres and are typically associated with genetic factors. The partial seizures are a local electrical discharge in the brain and are typically caused by brain injury, stroke, or tumor (9).
Glutamatergic synapses play a very important role in all epileptic phenomena. The enhanced activation of post-synaptic glutamate receptors (ionotropic and metabotropic) is proconvulsant. Antagonists of NMDA receptors and AMPA receptors are found to be potent anticonvulsants in many animal models of epilepsy (66). The animal models of epilepsy support the role of glutamate transporters in preventing seizure phenotypes. In a study, Wang and his colleagues showed that astrocyte-specific Tsc1 gene inactivation in mice (Tsc1 cKO mice) results in progressive epilepsy also the glutamate transporter (GLT-1 and GLAST) expression and function is impaired in Tsc1 cKO astrocytes. The study suggests that Tsc1 inactivation in astrocytes causes dysfunctional glutamate homeostasis which results in seizure development in tuberous sclerosis complex (TSC) (67). The role of glutamate has also been studied in epilepsy patient and the EEG and microdialysis studies revealed that the basal glutamate concentration in epileptogenic areas is 4.7 times higher than in non-epileptogenic areas of the hippocampus (68). The enhanced EAAT2 expression can protect against SE-induced death, neuropathological changes, and chronic seizure development which shows EAAT2 transporter to be a potential therapeutic target for epilepsy (69).
Stroke is a disease that affects the arteries leading to and within the brain. It is the No. 5 cause of death and a leading cause of disability in the United States. A stroke occurs when a blood vessel that carries oxygen and nutrients to the brain is either blocked by a clot or bursts (or ruptures) eventually causing cell death (70). Nearly 800,000 people in the United States have a stroke every year, with about three in four being first-time strokes. About 87% of strokes are classified as an ischemic i.e. stroke occurs when a clot or a mass blocks a blood vessel, cutting off blood flow to a part of the brain (70).
The excitotoxic effect of glutamate is the major contributor to the pathogenesis of a stroke (71). Ischemia causes the release of glutamate into the synaptic cleft and extracellular space and this excess glutamate results prolonged activation of ionotropic glutamate receptors (iGluRs). The overstimulation of iGluRs thereby activates intracellular signaling cascades producing excitotoxicity and cell death (72). Blocking glutamate release or inhibiting glutamate-mediated post-synaptic excitability diminishes neural degeneration in an animal model of stroke (73,74). The preservation glutamatergic synapses, in the frontal cortex against the temporal cortex, plays a significant role in sustaining cognition and protecting against dementia following a stroke (75). Enhancement of EAAT2 expression using ceftriaxone has proved to effective in several ischemia animal models which suggest that EAAT2 could be a great target for stroke (76,77).
Major depressive disorder
Major depression, also known as unipolar or major depressive disorder, is characterized by a persistent feeling of sadness or a lack of interest in outside stimuli. According to the Journal of the American Medical Association, the lifetime incidence of depression in the United States is more than 20-26% for women and 8-12% for men (78). Initially, the deficit in function or number of monoamines (the monoamine hypothesis) was the contributing factor to the pathology of depression. However, there is evidence that neurotrophic and endocrine factors also play a major role (the neurotrophic hypothesis) in major depressive disorders (MDD). Histologic studies, structural and functional brain imaging research, genetic findings, and steroid research all suggest a complex pathophysiology for MDD with important implications for drug treatment (79). Functional neuroimaging investigations of patients suffering major depressive disorder revealed greater response in the amygdala, insula, and dorsal anterior cingulate cortex and lower response in the dorsal striatum and dorsolateral prefrontal cortex in individuals with the major depressive disorder than in healthy subjects (80).
Glutamate and its receptors are implicated in the pathophysiology of MDD, as well as in the development of novel therapeutics for this disorder (81). Several studies have described the role of glutamatergic neurotransmission in individuals with mood disorders. Abnormal glutamate levels have been observed in the plasma, serum, and cerebrospinal fluid (CSF) of individuals with bipolar disorder (82). Various postmortem studies have shown increased glutamate levels and reduced expression of EAAT1, EAAT2, EAAT3, EAAT4 and glutamine synthetase subjects with mood disorders (83-86). Acute stress causes a significant increase in spontaneous (unstimulated) glutamate release and significantly enhance K+- and 4-AP-induced Ca2+-dependent glutamate release (87). Stress-effects on GLt-1/EAAT2 levels does not result from changes in hippocampal morphology but it reflects underlying neurochemical and molecular properties of the hippocampus in response to stress (88). In the tail suspension and novelty suppressed feeding tests, mice with reduced GLT-1 activity in the habenula exhibited a depressive-like phenotype and also exhibited increased susceptibility to chronic stress in the open-field test (89). Treatment with antidepressant agents may decrease the plasma glutamate levels in depressed individuals (90). As glutamate is the major excitatory amino acid neurotransmitter, targeting its uptake and enhancing EAAT2 expression can lead to a potential therapeutic advantage in major depressive disorders.
Addiction is a primary, chronic disease of brain reward, motivation, memory and related circuitry. Addiction affects neurotransmission and interactions within reward structures of the brain, including the nucleus accumbens, anterior cingulate cortex, basal forebrain and amygdala (91). Individuals addicted to drugs of abuse such as alcohol, nicotine, cocaine, and heroin experience positive reinforcing (rewarding) effects from these drugs and this rewarding effect lead to initiation and maintenance of the drug-taking habit (92). Thus, understanding the neurochemical mechanisms behind the rewarding effects of the drugs of abuse is very important in eliminating the addiction and allowing people to lead a healthy life.
Initially, dopamine and endogenous opioids were focused while conducting research related to addiction. However, glutamate is now the new player in addiction and it to regulates dopamine release in the nucleus accumbens, one of the cerebral structures of the reward system. The balance between glutamate and acetylcholine prevents up-regulation of the system and entry into addiction (93). Animal studies demonstrate that the reinstatement of drug seeking occurs due to an imbalance in glutamate transmission from the prelimbic cortex to the nucleus accumbens core (94). Decreased expression of EAAT2 is seen in rats withdrawn from cocaine self-administration, while treatment with ceftriaxone upregulates GLT1/EAAT2 and attenuates cue-induced cocaine reinstatement. This reinstatement is reversed by EAAT2 blockade in core but not a shell, and this further emphasizes the role of GLT1/EAA2 as a potential therapeutic target for cocaine relapse (95).
Glutamate is the very critical neurotransmitter and any alterations in expression of its transporters or dysfunction of transporters could lead to several neurodegenerative diseases because of glutamate excitotoxicity. Restoration of transporter function, mainly EAAT2 (as it is responsible for about 90% of glutamate clearance) can be targeted to treat different neurological disorders. EAAT2 could be regulated at transcriptional (e.g. ceftriaxone), translational (e.g. pyridazine derivatives) and functional level (e.g. spider extracts) depending upon the disease presentation and in what level disease has impacted EAAT2 expression (9). The number of marketed drugs for neurological disorders works by modulating neuronal target which is the reason for a number of side effects (96). Therefore, regulation of EAAT2, which is a non-neuronal target could lead to the development of more potent and efficacious therapeutic agents with minimal side effects. The small molecules activator of EAAT2/GLT1 are still under research and development and hold a great promise in the development of successful candidate against neurodegenerative disease.
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