Glutamate Transporters in Health and Disease

Abstract

Glutamate transporters, or excitatory amino acid transporters (EAATs), are key proteins that regulate the excitatory tone in the central nervous system (CNS) by clearing synaptic glutamate, maintaining extracellular glutamate concentrations low enough to prevent receptor desensitization and/or glutamate-mediated excitotoxicity. Dysregulation of the function and/or expression of the EAATs is implicated in several diseases, including epilepsy, stroke, traumatic brain injury, drug abuse disorders, neurodegenerative disorders, and neuropathic pain, among others. In this chapter, we will discuss the regulatory mechanisms of EAATs in health and disease states. We will discuss post-translational modifications, trafficking deficits, reverse transport, and other regulatory processes. We will also discuss current approaches on potential therapeutic strategies targeting these transporters for many neuropsychiatric diseases.

1. Introduction

Glutamate transporters, or excitatory amino acid transporters (EAATs), play a crucial role in regulating excitatory activity in the central nervous system (CNS). Thus, studying their regulation is essential for understanding health and diseased states. In physiological states, glutamate is involved in memory, learning, and other processes, and EAATs exert a tight control of the synaptic concentration of glutamate, through clearance into glial and neuronal cells. In diseased states, glutamate transporter function and/or expression can be dysregulated, leading to devastating effects in the CNS. Under conditions of prolonged glutamate activation, cells become overexcited by glutamate and go through degeneration and, ultimately, death, in a process called excitotoxicity [1]. Glutamate-mediated excitotoxicity has been shown to be involved in numerous conditions, such as ischemic stroke, epilepsy, and traumatic brain injury. Further, dysregulated levels of glutamate transporters that result in abnormal synaptic glutamate concentration are observed in several pathologies, such as drug abuse disorders, neurodegenerative disorders, and neuropathic pain, among others. In this chapter, we will discuss the regulatory mechanisms of EAATs in physiological states and the implication of glutamate transporter dysregulation in neurological and neuropsychiatric disorders.

2. Glutamate transporters in health

In the mammalian CNS, glutamate serves as the main excitatory neurotransmitter and is a critical signal for neural communication and plasticity. Once released into the synaptic cleft, glutamate promotes specific signaling pathways in post-synaptic neurons by interacting with ionotropic and metabotropic glutamate receptors, which initiate downstream signaling. As extracellular glutamate cannot be enzymatically degraded, glutamate must be removed from the synaptic cleft through glutamate transporters [2]. Therefore, EAATs are imperative for proper neuronal functioning, as they clear synaptic glutamate and maintain excitatory balance.

2.1 Subtypes and localization in the nervous system

There are two main classes of glutamate transporters: EAATs, which are dependent on an electrochemical gradient of sodium (Na⁺) ions, and the vesicular glutamate transporters (VGLUT-1-3) and cystine-glutamate antiporters (xCT), which are Na⁺-independent (Table 1). EAATs and xCT are both found in cell membranes; however, xCT has much lower expression than the EAATs, whereas vGLUTs are found in the membrane of glutamate-containing synaptic vesicles. In this chapter, we will focus on EAATs. For a review of the xCT system, see [3]; for a review of vGLUTs, see [4].

Glutamate transporter subtype-Human homolog	Glutamate transporter subtype-Rodent homolog	Gene	Cell type	Anatomic localization
EAAT1	GLAST	SLC1A3	Astrocytes, oligodendrocytes	Cerebellum, cortex, spinal cord; Also, in testis and bone
EAAT2	GLT-1	SLC1A2	Mainly astroglia	Throughout brain and spinal cord; Also, in liver
EAAT2b	GLT-1b	SLC1A2	Mainly astroglia, also expressed in neurons	Throughout brain and spinal cord; Also in intestine, kidney, liver, and heart
EAAT3	EAAC1	SLC1A1	Neurons (dendrites and axon terminals)	Hippocampus, cerebellum, striatum
EAAT4	EAAT4	SLC1A6	Neurons (Purkinje cells)	Cerebellum, hippocampus, and basal ganglia; Also, in placenta
EAAT5	EAAT5	SLC1A7	Neurons (photoreceptors and bipolar cells)	Retina; Also, in liver
VGLUT1	VGLUT1	SLC17A7	Neurons	Cerebral cortex, hippocampus, and cerebellum
VGLUT2	VGLUT2	SLC17A6	Neurons	Thalamus and brainstem
VGLUT3	VGLUT3	SLC17A8	Neurons	Cerebral cortex, hippocampus, striatum, and raphe nuclei
xCT	xCT	SLC7A11	Neurons and glia	Hippocampus, cortex, hypothalamus, and dentate gyrus

Table 1.

Glutamate transporters: nomenclature (human/rodent/genes), cell type and anatomic localization.

EAATs are classified into five subtypes (rat/human homolog/gene): GLAST/EAAT1/SLC1A3, GLT-1/EAAT2/ SLC1A2, EAAC1/EAAT3/ SLC1A1, EAAT4/SLC1A6, and EAAT5/SLC1A7 [2]. EAAT1 and EAAT2 are mainly localized in astrocytes, whereas EAAT3-5 are neuronal [5]. The main transporters responsible for the uptake of synaptic glutamate are astrocytic EAAT1 and EAAT2; thus, they are key for preventing the accumulation of synaptic glutamate an avoid excitotoxicity [6]. EAAT1 is predominantly expressed in astrocytes within the cerebellar Purkinje cell layer [7]. EAAT2 is the predominant subtype of glutamate transporter in the CNS [8] and is expressed in astrocytes, select presynaptic neurons, and oligodendrocytes within the brain and spinal cord [9]. EAAT2 expression contributes to around 95% of total glutamate transport activity and represents approximately 1% of the total brain protein in the CNS [10], therefore, playing a key role in the maintenance of extracellular glutamate homeostasis. While both EAAT1 and EAAT2 are expressed within the same astrocytic plasma membrane, they are regulated differently; exogenous glutamate levels affect the cell-surface expression of EAAT1, but not EAAT2; EAAT2 is regulated by neuronal soluble factors, unlike EAAT1 [11]. Several splice variants of EAAT2 have been identified, with EAAT2b (or GLT-1b) being the most extensively studied. However, apparent functional differences between each of the variants are not readily apparent [12]. EAAT3 exhibits ubiquitous expression in the brain, playing a crucial role in regulating local glutamate concentrations, as it is predominantly located within post-synapsis, which allows for the buffering of nearby glutamate receptors, influencing excitatory neurotransmission and synaptic plasticity [13]. EAAT4, which is expressed in cerebellar neurons, is primarily responsible for maintaining low extracellular glutamate levels and preventing neurotoxicity in the cerebellum, together with glial EAAT1 [14]. EAAT5, a neuronal transporter mainly expressed in retina, plays a vital role in controlling glutamate release and mediating light responses in depolarizing bipolar cells in the retina [15].

See Figure 1 for an overview of the localization of EAATs in the CNS.

Figure 1.

Localization of neuronal and astrocytic excitatory amino acid transporters (EAATs) in the CNS. A. Overview of a glutamate tripartite synapse (pre- and post-synaptic neurons and astrocytes) showing the localization of astrocytic transporters EAAT1 and EAAT2 and neuronal transporter EAAT3, in the cerebrum and hippocampus. After glutamate (Glu, red dots) is released from the presynaptic terminal and stimulates postsynaptic glutamate receptors AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic) and NMDA (N-methyl-D-aspartate), and metabotropic glutamate receptors (mGluRs), it undergoes reuptake through EAATs present in astrocytes and neurons. In astrocytes, glutamate is converted into glutamine (Gln, green dots), which is shuttled back to neurons. In addition to glutamate, EAAT3 also transports the glutathione precursor cysteine (not shown) into neurons, which is required to produce glutathione. B. Overview of a glutamate tripartite synapse (pre- and post-synaptic neurons and astrocytes) showing the localization of astrocytic transporters EAAT1 and EAAT2 and neuronal transporters EAAT3 and EAAT4 in the cerebellum. Note that EAAT4 is only expressed in synapses in the cerebellum. C. Overview of a glutamate tripartite synapse (pre- and post-synaptic neurons and astrocytes) showing the localization of astrocytic transporter EAAT1 and EAAT2. Note that EAAT2, in the retina, is mainly expressed in neurons such as photoreceptors and bipolar cells, and neuronal transporter EAAT5 is only expressed in bipolar cells neurons in the retina. EAAT5 is restricted to. Created with BioRender.

2.2 Stoichiometry and structure of the EAATs

EAATs function through an electrogenic process that is dependent on the co-transport of three Na⁺ (sodium) ions and one proton with glutamate and the counter-transport of one K⁺ (potassium) ion out of the cell, in addition to an uncoupled Cl⁻ (chloride) current (Figure 2A) [16, 17]. This process is also known as an “induced fit mechanism,” as Na⁺ binding is required for glutamate binding, which also contributes to the high selectivity of EAATs for glutamate [18]. As EAATs are secondary active transporters, the ionic gradients of these substrates facilitate the glutamate transport cycle. EAATs rely indirectly on Na^+/K⁺-ATPase (NKA) to generate these ion gradients [19].

Figure 2.

Molecular properties of EAATs. A. Schematic displaying substrate stoichiometry associated with glutamate transport, displayed as trimer in the membrane. Glutamate (Glu) uptake is driven by co-transport of three Na⁺ ions and one proton (H⁺) and by counter-transport of one K⁺ ion. EAATs also show an uncoupled Cl⁻ conductance (shown as dotted arrow). B. Transmembrane topology of glutamate transporters consisting of eight transmembrane domains and two hairpin loops (HP1 and HP2). Transmembrane domains are shown in dark blue, and scaffold domains in light blue. C. Glutamate transport cycle of a single protomer. Transport domain (magenta) of protomer moves along the scaffold domain (green) to go from an outward-facing conformation (far left) to an inward conformation (far right), passing through an intermediate conformation in which chloride conductance can occur. Created with BioRender.

EAATs are transmembrane integral proteins that traverse the cell membrane eight times (Figure 2B). EAATs consist of three protomers that can independently transport glutamate [16]. They have four key domains: a scaffold domain, or trimerization domain (comprised of transmembrane domains 1, 2, 4, 5), which remains stationary; a transporter domain (transmembrane domains 3, 6, 7, 8) that moves as a large rigid body along the scaffold domain in a twisting elevator-like motion to transport glutamate and its cosubstrates into the cell, and two hairpin domains (hairpin domains 1 and 2) that act as intracellular and extracellular gates [20]. These transporters follow an “alternating-access model” through this motion, which brings the transporter from an outward-facing conformation, where glutamate and cosubstrates bind, to an inward-facing conformation, where they are released (Figure 2C) [21]. While transporters and ion channels have historically been viewed as distinct proteins, EAATs also function as anion channels, which open during an intermediate state of the transport cycle [22]. This anion channel in EAATs is selective for chloride and is stoichiometrically uncoupled from glutamate transport [23]. Additionally, this chloride conductance may mediate neuronal excitability and ion homeostasis [24]. The malfunction of this chloride channel has also been linked to neurological diseases such as episodic ataxia [25]. Recent cryo-EM studies have reported the structures of EAAT1 [26], EAAT3 [27], and EAAT2 [28, 29] in several states in the presence of substrate glutamate or inhibitors. These studies are highly relevant as they revealed the structural basis of coupled substrate and ion binding.

2.3 Modulators of the activity of EAATs

In the last decades, compounds that modulate EAAT functions, directly or indirectly, have been identified and developed (some examples are outlined in Table 2).

Compound	Mechanisms	References
Indirect activators
Riluzole (2-amino 6-(trifluoromethoxy)benzothiazole)	Increases activity and expression of EAAT2 Blocks sodium channels	[30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43]
Positive allosteric modulators (PAMs)
Parawixin1 (unknown chemical structure)	Activates EAAT2 activity	[44]
GT949 (3-((4-Cyclohexylpiperazin-1-yl) (1-phenethyl-1H-tetrazol-5-yl) methyl)-6-methoxyquinolin-2(1H)-one and GT951 6-methoxy-3-((1-phenethyl-1H-tetrazol-5-yl) (4-(3-(trifluoromethyl) phenyl) piperazin-1-yl) methyl) quinolin-2(1H)-one)	EAAT2 PAMs, activity, no effect on NMDA-mediated currents In vitro neuroprotection in glutamate-mediated excitotoxicity models	[45, 46]
Parawixin10 (S)-N1-(3-(2-amino-5-guanidinopentanamido)propyl)-N4-(3-(2-(4-hydroxy-1H-indol-3-yl)acetamido)propyl)-N1,N1,N4,N4-tetramethylbutane-1,4-diaminium iodide)	Increases glutamate uptake in rat brain synaptosomes. PAM of EAAT1 and EAAT2 Offers neuroprotection in in vitro stroke models and in vivo epilepsy models	[47, 48, 49, 50]
[(R)-AS-1] [(R)- N-Benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide)]	Selective EAAT2 PAM Offers in vivo protection from seizures	[51]
Competitive Inhibitors
DL-TBOA and analogs (DL-threo-beta-benzyloxy aspartate)	Non-transportable blocker of all subtypes of EAATs	[52, 53, 54, 55, 56, 57, 58, 59, 60]
TFB-TBOA [(3S)-3-[[3-[[4-(Trifluoromethyl)benzoyl] amino] phenyl] methoxy]-L-aspartic acid]	Selective inhibitor of EAAT1 and EAAT2	[61, 62, 63, 64]
Non-competitive inhibitors
Several putative negative allosteric modulators	Selective and non-selective inhibition of EAAT1, EAAT2, and/or EAAT3	[65]

Table 2.

Examples of modulators of excitatory amino acid (EAAT) activity.

The table includes indirect activators (compounds that interact indirectly with EAATs augmenting their catalytic activity, usually acting through multiple mechanisms), positive allosteric modulators (PAMs, compounds that interact directly with EAATs at an allosteric site and putatively increase their activity), competitive inhibitors (compounds that bind to the same binding sites as glutamate, competing with glutamate for binding to the transporter protein; some of these inhibitors are substrates themselves, some are exchanged with internal glutamate and induce glutamate release), and non-competitive inhibitors (or negative allosteric modulators, NAMs, that act by binding EAATs at an allosteric site, often leading to a conformational change that impacts transporter dynamics). In addition, we provide some of their known mechanisms and include further references.

Several compounds indirectly interact with EAATs, augmenting their catalytic activity, usually acting through multiple mechanisms, such as riluzole [30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 66]. Inhibitors of EAAT activity are grouped into competitive (bind to the same binding sites as glutamate) and non-competitive (bind somewhere else, i.e., to an allosteric site). Some of these inhibitors are substrates themselves, and some are exchanged with internal glutamate, thereby inducing glutamate release [52].

An emerging approach to drug design focuses on allosteric modulators, which bind to allosteric sites and alter the conformation of the orthosteric binding site, affecting transport by either enhancing (positive allosteric modulation, PAM) or inhibiting (negative allosteric modulation, NAM) binding affinity and transport. Allosteric modulation may offer advantages such as targeted drug therapy and increased specificity, thus offering promising prospects for drug discovery and the development of novel therapeutics [67]. Recently, allosteric sites have been described in EAATs [45, 46]. The development of selective EAAT PAMs and NAMs may be useful tools to decipher how drugs can affect the transport cycle and to investigate the intrinsic properties and functions of the EAATs [68].

Further research on allosteric modulators of EAATs is necessary to address several outstanding questions. These include determining the precise location and physiological significance of the allosteric binding sites, investigating whether these compounds can stabilize specific conformations of the transporter, understanding how different conformations of EAATs transmit signals into the cell, and exploring the possibility of allosterically modulating transporter-mediated efflux. Ultimately, this knowledge could be invaluable for advancing our understanding of EAAT modulation and for informing the drug development process [69].

2.4 Modulators of the expression of EAATs

The expression of EAATs is altered in certain disease states, leading to changes in neuronal excitation. This regulation can occur through transcriptional and translational processes [70]. However, the exact mechanisms underlying EAATs expression regulation are not well understood; hence, synthetic modulators of expression could serve as valuable tools for investigating the functions and regulation of EAATs in both physiological and disease contexts. There are exogenous drugs, endogenous molecules, and proteins that increase EAATs expression (transcriptional and level, i.e., increase gene transcription, and translational, i.e., increase the protein expression) and inhibitors of EAAT expression. Additional research is needed to evaluate whether a compound that enhances GLT-1 expression and has good brain penetrance, favorable pharmacokinetic properties, and low risk of side effects and toxicity can be developed into a therapeutic drug [71]. Some commonly studied EAAT expression modulators are outlined in Table 3.

Compound	Mechanism	References
Expression enhancers
Ceftriaxone ((6R,7R)-7-{[(2Z)-2-(2-amino-1,3-thiazol-4-yl)- > 2-(methoxyimino) acetyl]amino}MI-3-{[(2-methyl-5,6-dioxo-1,2,5,6-tetrahydro-1,2,4-triazin-3-yl)thio]methyl}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid)	β-Lactam antibiotic Selective enhancer of EAAT2 expression, through transcriptional activation via mechanisms involving PI3K/Akt/NF-κB	[72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100]
Clavulanic acid ((2R,5R, Z)-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-aza-bicyclo [3.2.0] heptane-2-carboxylic acid)	Structural analog β-lactam but lacks antibiotic effects Increases EAAT2 expression (has better oral availability and blood-brain barrier penetration than ceftriaxone)	[77, 79, 101, 102, 103, 104, 105, 106]
LDN/OSU-0212320 (Thiopyridazine and pyridazine derivatives)	Increases EAAT2 expression through translational activation	[107, 108, 109, 110, 111, 112]
Amitriptyline (3-(10,11-dihydro-5H-dibenzo [a, d] cycloheptene-5-ylidene)-N, N-dimethylpropan-1-amine)	Tricyclic antidepressant Induces EAAT2 expression and ameliorates neuropathic pain	[113, 114]
N-acetylcysteine ((2R)-2-acetamido-3-sulfanylpropanoic acid))	Increases EAAT2 expression- Reduces drug-seeking/taking behavior for cocaine, nicotine, and ethanol	[115, 116, 117, 118, 119, 120, 121]
Minocycline ((2E,4S,4aR,5aS,12aR)-2-(Amino-hydroxy; -methylidene)-4,7-bis(dimethylamino)-10,11,12a-trihydroxy-4a,5,5a,6-tetrahydro-4H-tetracene-1,3,12-trione)	Broad-spectrum tetracycline antibiotic Ameliorates downregulation of GLT-1 expression in neuropathy model	[122]
Expression inhibitors
PMA (phorbol 12-myristate 13-acetate)	Activator of PKC Decreases the activity and expression of GLT-1 through clathrin-mediated endocytosis (Also has actions on EAAT3)	[123, 124, 125, 126, 127, 128]
Synthetic cathinone MDPV (3,4-methylenedioxypyrovalerone methylenedioxy pyrovalerone)	Downregulates GLT-1 expression, and norepinephrine-dopamine reuptake	[129]
Amphetamine (1-phenylpropan-2-amine)	Potent CNS stimulant Downregulates EAAT3 expression Enters dopamine neurons via DAT and triggers endocytosis of EAAT3, in a process involving dynamin- and Rho-mediated mechanisms	[130, 131]

Table 3.

Examples of modulators of Excitatory Amino Acid Transporters (EAAT) expression.

The table includes examples of drugs that enhance EAATs expression (at the transcriptional and translational level), and examples of drugs that inhibit EAATs expression, along with some of their known mechanisms and further references.

2.5 Trafficking and post-translational modifications of EAATs

The expression and function of EAATs are regulated at the genetic, epigenetic, transcriptional, post-transcriptional, and translational levels [132]. Dysregulation of these processes can lead to severe outcomes such as high levels of extracellular glutamate and excitotoxicity [6]. The activity of transporters can be regulated by many means, such as ubiquitination, phosphorylation glycosylation, and sulfhydryl oxidation, among others [133, 134, 135].

Post-translational modification of GLT-1 by ubiquitin conjugation to lysine residues has been found to mediate the transporters constitutive internalization and degradation. This regulated clathrin-mediated endocytosis in basal conditions determines the availability of transporter on the cell surface and therefore relates directly to activity levels and rate of glutamate transport [123, 136]. Additionally, the palmitoylation of GLT-1 has been shown to drive glutamate uptake kinetics. Palmitoylation involves the covalent attachment of palmitic acid to one of more cysteine residues of the target protein. Studies suggest that palmitoylation of GLT-1 is important for glutamate uptake capacity, as a reduction in palmitoylated GLT-1 leads to impairments in glutamate clearance [137]. The sumoylation of GLT-1 in physiological signaling has also been identified, which involves the attachment of a SUMO protein to a lysine residue on the target protein and was found to regulate GLT-1 subcellular localization [138]. Notably, several groups have reported aberrations in the constitutive trafficking, expression, and activity levels of glutamate transporters due to alterations in post-translational modifications in many disease states, which will be further discussed in Section 3.

3. Glutamate transporters in disease

Dysregulation of EAATs plays a significant role in many neuropsychiatric diseases/disorders. Below, we briefly discuss two mechanisms of EAAT dysregulation involved in several disorders: glutamate efflux via transporter reversal and downregulation of the expression of glutamate transporters. We also briefly describe studies of knockout of EAAT that were pivotal for our understanding of the role of glutamate transporters in health and disease.

3.1 Glutamate transporter reversal

Under physiological conditions, the direction of glutamate transport is inward; however, in pathological conditions, the function of Na^+/K⁺-ATPase (NKA) becomes dysfunctional, with subsequent disruption of Na⁺/K⁺ electrochemical gradient needed for glutamate translocation, resulting in increases in extracellular K⁺ and decreases in Na⁺ decreases, which leads to glutamate transport in the outward direction [139]. In support of this, massive increases in extracellular K⁺ and indiscriminate release of glutamate have been reported following concussive and lateral fluid percussion brain injuries [140], ischemia [141], and oxidative stress in a SOD1 mutation amyotrophic lateral sclerosis (ALS) model [142, 143].

It remains to be clarified whether EAAT functional activators or expression enhancers will facilitate glutamate clearance under excitotoxic conditions or will intensify reverse transport. However, previous preclinical studies suggest neuroprotective properties of these compound classes against ischemia, which encourages future drug development using this strategy. Nevertheless, as glutamatergic signaling plays crucial roles in brain development, cell survival, and synaptogenesis, these pharmacological strategies encounter similar challenges to other drugs targeting glutamate-mediated mechanisms, such as the potential for significant adverse effects [71, 144].

3.2 Glutamate transporter downregulation

Reduced expression and function of EAATs has been reported in numerous neurological disorders. Although the exact mechanism of downregulation has yet to be fully established, it results in impairment of the overall function of glutamate transporters, which plays an important role in the etiology of neurological diseases (see below).

For an overview of the role of EAATs at glutamatergic synapses at physiological (A) and pathological (B) states, see Figure 3.

Figure 3.

Tripartite glutamatergic synapse in physiological and pathological conditions. Schematic representing glutamatergic transmission in the context of physiological (A) and pathological (B) states that are associated with glutamate-induced excitotoxicity and EAAT2 protein dysregulation. A. In B. Hyper-glutamatergic signaling results in excess glutamate (red dots) release, overactivation of ionotropic glutamate receptors AMPA and NMDA, and aberrant calcium (blue dots) influx leading to the activation of various cell death pathways. In many disease states, glutamatergic dysfunction can lead to transporter reversal (shown by the arrow pointing to glutamate efflux from the astrocytes to the extracellular environment) and downregulation (shown as lower EAAT2 expression than in A), further exacerbating excitotoxic outcomes, such as in ischemic stroke. Na+/K+ ATPase (NKA) dysfunction also contributes to the dysregulation of EAAT activity. Downregulation of EAAT2 expression is also observed in pathologies such as in drugs of abuse disorders (depicted in the figure as drug-seeking behavior) and neuropathic pain. Created with BioRender.

3.3 Studies with knockout of EAATs

Studies with knockout of glutamate transporters reveal a major role for EAATs in clearance of glutamate, excitotoxicity, and associated neurotransmission. A key study by Tanaka’s group in the 1990s demonstrated that EAAT2 gene knockout resulted in lethal spontaneous seizures and increased susceptibility to acute cortical injury in mice [145]. Later studies confirmed severe disturbances in mice lacking GLT-1 and GLAST [146], including elevated extracellular glutamate levels, exacerbated hippocampal neuronal damage after brain injury [147], impairment of several essential aspects of neuronal development [148], neurodegeneration and progressive paralysis [149]. Mice lacking EAAT1 have decreased cerebellar function, reduced motor coordination, hearing loss, and disturbed retinal function. Antisense knockdown of GLAST compromises retinal function [150], and constitutive deletion of GLAST results in markedly reduced alcohol consumption and preference [151]. Mice lacking EAAT3 develop dicarboxylic aminoaciduria (Kegg’s disease), exhibit reduced spontaneous locomotor activity, and may age prematurely [152]. Additionally, they experience depletion of glutathione and neuronal cell loss [153], suggesting that EAAT3 plays a critical role in providing cysteine for glutathione synthesis [154]. Humans lacking EAAT3 develop dicarboxylic aminoaciduria, a rare metabolic disorder that causes the body to excrete too much aspartate and glutamate in urine, and human EAAT3 polymorphisms have been reported to be associated with obsessive-compulsive disorders [155]. In EAAT4 knockout mice, Purkinje cells are more likely to die [156], and in a double knockout of EAAT1 and EAAT4, both highly expressed in the cerebellum, a differential effect was observed in the spontaneous firing pattern and survival of Purkinje cells [14], demonstrating the essential role of these transporters in the cerebellum. Finally, EAAT5 was shown to shape the retinal light responses, in an EAAT5 knockout model [157].

3.4 Stroke

Stroke is the third leading cause of death in the United States, accounting for 700,000 fatalities each year [158]. Following an ischemic event, inadequate blood flow to the brain prevents the delivery of oxygen and glucose. The deprivation of these substrates to neurons causes cell death and lasting brain damage [159]. Following a focal ischemic event, the resulting damage manifests into two distinct regions that are classified as the infarction core and the penumbra. The core is the region of the brain in which the primary occlusion occurs, which undergoes rapid and irreversible cellular death within minutes of ischemic onset, usually due to necrosis [160]. The penumbra is classified as the region that surrounds the core, which receives reduced blood supply but remains partially metabolically active and therefore contains salvageable tissue. However, this region is at risk of cell death if blood flow is not quickly restored [159], making the penumbra the primary area of interest for new targeted therapies.

Glutamatergic dysfunction is a key factor contributing to neuronal cell death following ischemic stroke [161, 162]. The lack of oxygen due to cessation of blood flow results in depleted ATP stores, thereby disrupting the ionic gradients responsible for regulating neuronal firing, resulting in increased action potentials and aberrant glutamatergic signaling. Excessive release of glutamate during ischemia leads to overactivation of postsynaptic ionotropic glutamate receptors. Released glutamate may also diffuse out of the synaptic cleft, causing activation of distant (extrasynaptic) receptors and subsequent elevated calcium influx into the cytosol. This affects calcium-sensitive organelles such as the mitochondria and endoplasmic reticulum [163] and causes the release of calcineurin and calpains, mediators of cell death [164]. Mitochondrial dysfunction and oxidative stress also play key parts in the excitotoxic phenotype [165]. Thus, minimizing glutamate-induced excitotoxicity by regulating the aberrant signaling cascade following stroke can be therapeutically beneficial in improving post-stroke outcomes.

As EAATs are responsible for removing glutamate from the synaptic space to help end neurotransmission, these proteins play a key role in mitigating excitotoxic outcomes. Recent research has focused on better understanding the regulation of EAATs during or after an ischemic event as well as on strategies to modulate their expression and/or activity to bolster glutamate clearance and promote cell recovery. Many groups have identified temporal and spatial alterations in glutamate transporter expression over the course of ischemic injury [166]. Additionally, the activity and function of glutamate transporters can be significantly affected by the severity of ischemic insult, with severe insults even causing a reversal in glutamate transport due to disruptions in the ionic gradients that drive these transporters [167]. Membrane translocation of glutamate transporters after ischemia has also been suggested, with elevations in glutamate release causing rapid changes in the diffusion and clustering of EAAT2 along the plasma membrane [168]. Taken together, the regulatory movement and response of glutamate transporters to ischemic insult is complex and multifaceted.

An extensively researched pathway in addressing ischemic injury involves the modulation of glutamate transporter expression. Pharmacological preconditioning with several β-lactam antibiotics, most notably ceftriaxone, an EAAT2 expression enhancer, has shown promising results as a treatment option in several neurological disorders including ischemic stroke. Previous work has found that ceftriaxone, when administered 48 hours before OGD, reduced neuronal death by 20–50%, thereby providing neuroprotection [72]. In this model, daily administration of ceftriaxone 5 days prior to middle cerebral artery occlusion (MCAO) provided neuroprotection through a reduction in infarct volume as well as neuroinflammatory and apoptotic factors through EAAT2 upregulation [73]. Additionally, a study found that daily administration of ceftriaxone as well as N-acetylcysteine 5 days prior to focal cerebral ischemia also significantly increased EAAT2 expression levels in addition to a reduction of infarct volume [115]. Other compounds that protect neurons from glutamate-induced excitotoxic death, such as LDN-OSU0212320, decrease infarct volume in mice when administered 24 hours prior to photothrombotic ischemia; however, only in male mice, suggesting that this treatment may not be effective in the female population and further emphasizing the importance of sex differences in targeted treatments [107, 108]. A significant drawback associated with β-lactam antibiotics in enhancing EAAT2 expression is the extended time required for drug onset (≥24 hours) [169]. Therefore, in the context of clinical ischemic injury, more fast-acting compounds need to be developed. EAAT PAMs offer a different approach to restoring glutamate clearance through the enhancement of glutamate uptake; however, this hypothesis remains to be tested in animal models of stroke.

3.5 Traumatic brain injury

Studies on traumatic brain injury (TBI) in both humans and animals have shown an acute increase in tissue glutamate concentrations that persist at elevated levels for up to 5 days in humans. This suggests a delay or insufficient glutamate clearance by glutamate transporters following TBI [71, 170]. Many studies have established that the subsequent glutamate-mediated excitotoxicity plays a significant role in acute post-injury neurodegenerative events [171]. In addition, decreased GLT-1 expression was shown in several TBI preclinical studies [147], which is consistent with the decreased EAAT2 activity observed in TBI patients [171]. Furthermore, antisense knockdown of GLT-1 in rat aggravates neuronal damage following TBI [172].

TBI is a complex pathology of many etiologies, which varies depending on the severity of the injury and the location of the affected brain tissue. In cases of concussion, where brain injury is typically reversible and symptoms resolve over time [173], the opportunity to reduce acute glutamate excitotoxicity by targeting GLT-1 offers potential for neuroprotection. However, for other types of TBI, additional pharmacological approaches may be necessary to address the diverse pathophysiological mechanisms involved [174].

3.6 Epilepsy

Epilepsy is a group of disorders characterized by recurrent spontaneous seizures that appear to stem from intricate processes involving various neurotransmitter systems, including glutamate [175, 176]. This leads to an imbalance between neuronal excitatory and inhibitory activities, ultimately culminating in epileptogenesis [177]. Numerous drugs are available as anti-seizure medications; however, none of the current treatments are considered disease-modifying, as they only suppress seizures without addressing the development and progression of epilepsy.

As previously mentioned, a key preclinical study revealed that mice lacking GLT-1 are prone to exhibit seizures [145]. Additionally, conditional deletion of GLT-1 revealed that GLT-1 protects against fatal epilepsy [178]. Loss of GLAST or GLT-1 led to elevated extracellular glutamate levels, neurodegeneration, and progressive paralysis, and loss of EAAC1 caused mild neurotoxicity and resulted in epilepsy [149]. In patients with medial temporal lobe epilepsy (mTLE), increased extracellular glutamate levels were observed [179] as well as decreased levels of EAAT1, EAAT2, and EAAT3 [180], reviewed in Ref. [176]. Decreased levels of GLT-1 were also observed in animal models of epilepsy such as pilocarpine-induced [181], albumin-induced [182], tuberous sclerosis-induced [183], FeCl₃-induced models [184], and chest compression-induced audiogenic model [185].

However, some studies did not observe changes in EAAT2 levels in patients [186] and in animal models including kindling [187], anticonvulsant ketogenic diet [188], and spontaneously epileptic rats [189], suggesting that EAAT2 may be implicated in the etiology of only some types of epilepsy. Another concept suggests that a deficiency in glutamine synthetase in astrocytes may be the molecular mechanism underlying extracellular glutamate accumulation and seizure generation [190].

Nonetheless, the upregulation of EAAT2 via transcriptional and translational regulation has demonstrated success in vivo by reducing spontaneous recurrent seizures and providing neuroprotection [191].

Parawixin10, a compound isolated from Parawixia bistriata spider venom, is a non-selective PAM of EAAT1 and EAAT2, with in vitro neuroprotective properties [47] and in vivo neuroprotection in epilepsy models of intrahippocampal injection of NMDA [48], kainic acid and pentylenetetrazol (PTZ) [49], and pilocarpine [50]. These studies served as proof of concept that EAAT1-2 PAMs may offer neuroprotection and anticonvulsant properties. More recently, the selective EAAT2 PAM [(R)-AS-1] revealed favorable anticonvulsant and safety profiles, and significant protection in mice against seizures in acute and chronic animal models of seizures [51]. These novel compounds may have disease-modifying potential in acquired epilepsy, which will require future experimental testing.

Several studies demonstrated mutations in EAAT1 are implicated in episodic ataxia 6 (EA6), a chronic condition characterized by epilepsy, nystagmus, and tinnitus. These mutations result in impaired glutamate uptake and alterations in anion conductance; however, how exactly these mutations affect EAAT1 expression, subcellular localization, function, and the complex neurological phenotype of EA6 remains to be understood [25].

Collectively, these studies suggest that astrocytic glutamate uptake plays a critical role in protecting neurons from hyperexcitability. However, discrepancies in some findings indicate that the exact mechanisms remain elusive. Nevertheless, the idea that modulating astrocytic EAATs represents a potential therapeutic approach to provide neuroprotection, to prevent spontaneous recurrent seizures, and to halt epileptogenesis [192].

3.7 Pain

Pain, an aversive sensory experience arising from actual or perceived tissue damage, constitutes a physiologic response to noxious stimuli or disease, serving as a protective mechanism to prompt seeking care or preventing further harm [193]. Pain perception involves complex interactions among cellular and molecular components, including neurons, glia, glutamate receptors, and transporters that utilize glutamate, the primary transmitter released by sensory afferents in the nervous system [194, 195, 196]. However, when pain extends beyond the acute injury phase, persisting for more than 3 months, it transitions into a chronic disease. Chronic pain, a prevalent motive for medical intervention, correlates with heightened risks of poor mental health, opioid dependency, and diminished quality of life.

During chronic pain development, excess glutamate released in the peripheral and central nervous system contributes to elevated extracellular glutamate levels, which overactivates glutamate receptors and exacerbates pain symptoms. Prolonged overactivation leads to neuroplasticity in pain pathways, amplifying pain signals, a phenomenon known as central sensitization [197]. Glutamate transporters, particularly EAAT2, counteract this process by removing extracellular glutamate and reducing pain signaling [196, 198]. In the pain pathway, EAAT2 is expressed in the anterior cingulate cortex, somatosensory cortex, hippocampus, and dorsal horn of the spinal cord [199].

After injury occurs, the expression of EAATs changes, potentially contributing to chronic pain conditions. Numerous pain models have been used to study EAATs during neuropathic pain development but with varying results. For example, in nerve-injury models of pain, studies found an initial increase in EAAT2 3–7 days after surgery, followed by a steep downregulation below baseline [200]. Other studies did not observe this initial upregulation in EAAT1 or EAAT2 early after injury and instead observed an upregulation of EAAT3 and a downregulation of EAAT1 and EAAT2 [101]. Because of these inconsistencies, the relationship between neuropathic pain development and EAAT expression remains unclear.

Various mechanisms are being explored for chronic pain therapy, and targeting the glutamatergic system to reduce pain transmission shows promising potential [196, 201]. Although glutamate receptor antagonism provides anti-nociception, it may be associated with severe side effects, including sedation, hallucinations, and cardiac instability, limiting its use in outpatient settings [202]. A potential novel therapeutic approach involves the modulation of astrocytic EAATs, located on cells that surround glutamatergic neurons, aiming to restore homeostasis by lowering the concentration of extracellular glutamate. In this sense, downregulation or inhibition of EAATs has been reported to increase pain [203], whereas increased EAAT2 expression can mitigate pain [204, 205, 206]. For example, overexpression of GLT-1 in the spinal cord attenuated the induction of inflammatory and neuropathic pain, suggesting this could be a strategy for pain [207]. Furthermore, positive allosteric modulation of EAATs can enhance their efficiency in various neurological diseases, offering neuroprotection, and is a potential target for chronic pain.

In preclinical pain models, certain drug classes have been shown to have anti-nociceptive properties by modulating EAATs through the enhancement of EAAT expression and removal of excessive glutamate [196, 203, 208]. Some of these drugs include β-lactam antibiotics (such as ceftriaxone), β-lactamase inhibitors (such as clavulanic acid), tetracycline antibiotics (such as minocycline), anticonvulsants (including VPA and riluzole), and tricyclic antidepressants (such as amitriptyline) [196]. Ceftriaxone has undergone extensive research as a potential therapy for modulating EAAT2 expression. Studies in the chronic constriction injury (CCI) pain model demonstrated that ceftriaxone upregulated GLT-1 expression and glutamate uptake in the spinal dorsal horn and resulted in anti-nociceptive effects [74, 75]. Furthermore, its impact extended to a model of multiple sclerosis, where ceftriaxone not only reversed tactile allodynia but also halted the progression of motor weakness and paralysis. Notably, in both models, ceftriaxone reversed the reduction in EAAT2 expression and astrocyte activation in the lumbar spinal cord, suggesting its potential to suppress glial activation and alleviate pain [75].

Concerns about the antibiotic activity of ceftriaxone led to the investigation of clavulanic acid, which is devoid of antibiotic properties. In the CCI model, both clavulanic acid and ceftriaxone demonstrated anti-nociception, increased EAAT2 expression in the rat spinal cord, and reversed EAAT2 downregulation at day 14 post-surgery [209]. Clavulanic acid may be a candidate for relieving pain in diabetic peripheral neuropathy, and its benefits could be attributed to increased EAAT2 expression [101].

Another approach to modulate EAAT2 that could be beneficial for various pain conditions is through positive allosteric modulation (PAM), which involves the enhancement of extracellular glutamate uptake. Several preclinical pain studies demonstrated a downregulation of EAAT2, thus enhancing the activity of the remaining transporters via PAM represents a promising new avenue for future drug development [196, 210]. Selective EAAT2 PAMs will restore glutamate homeostasis by enhancing the uptake of excessive glutamate, rather than blocking glutamatergic transmission; thus, this approach is expected to be safer and devoid of the dissociative effects observed with NMDA receptor antagonists [196]. Additionally, EAAT2 PAM compounds differ significantly from transcriptional or translational upregulation of EAAT2 expression, such as ceftriaxone, which display varying efficacies across individuals, raising concerns about safety, efficacy, and adverse effects. EAAT2 PAMs work directly on the transporter, rapidly increasing glutamate uptake efficiency, resulting in a quicker onset compared to drugs that modulate expression. They are highly selective, providing a more robust and safe clinical profile compared to epigenetic-modifying expression enhancers that are often associated with adverse side effects [196, 211]; however, further studies are needed to confirm these expectations.

3.8 Substance use disorders

The maintenance of low extracellular glutamate levels in the CNS is important for proper cognitive functions such as learning and memory [212]. Both learning and memory are involved in the cycle of addiction, as is evident by relapses [213]. Substance use disorder is characterized by compulsion to use a substance, inability to limit intake of that substance, and the appearance of negative affect following the use of the drug. Regions such as the prefrontal cortex, amygdala, and hippocampus, which are involved in learning and memory, have projections connecting them to key regions involved in addiction, such as the ventral tegmental area and nucleus accumbens [214]. Drug use activates glutamatergic neurotransmission in the mesocorticolimbic system [215]. Following repeated use, drug-specific changes occur in these brain regions that drive the susceptibility to relapse and chronic drug use [216]. Furthermore, in abuse models, dysregulation of glutamate levels is observed in key brain regions associated with addiction, such as the prefrontal cortex, hippocampus, and nucleus accumbens [217]. All drugs of abuse disrupt glutamate homeostasis during use, increasing synaptic glutamate release. Numerous studies have demonstrated the importance of glutamatergic signaling in the nucleus accumbens on drug-seeking [218]. The importance of changes in glutamatergic tone that occur in addiction is further evident as pharmacologically restoring glutamate levels reduces drug-seeking behavior [219].

Targeting EAAT2 specifically has displayed promising results in the context of addiction. The use of drugs such as cocaine, cannabinoids, amphetamine, ethanol, nicotine, and opiates results in significant changes in EAAT2 levels [216]. Preclinical studies have shown that ceftriaxone inhibits cue- and drug-induced reinstatement seeking for cocaine [220], heroin [221], methamphetamine [222], and nicotine [223]. Additionally, administration of ceftriaxone attenuates the development of cocaine-induced conditioned place and prevents the decrease in EAAT2 expression in the nucleus accumbens [224]. Ceftriaxone also reduces alcohol intake [76]. Clavulanic acid reduces the reinforcing efficacy of cocaine and reduces cocaine-conditioned place preference [77]. Additionally, clavulanic acid reduced morphine-conditioned place preference, morphine-induced hypothermia, and locomotor sensitization [102]. Riluzole prevents cocaine reinstatement while restoring the expression of EAAT2 in the nucleus accumbens [225]. However, clinical trials found that riluzole was not effective for cocaine use disorder, when taken during the period of cocaine dependency [226]. Interestingly, riluzole has shown some promising effects in clinical trials for methamphetamine dependence, as it decreased symptoms, such as craving, withdrawal, and depression, experienced by men in an outpatient setting [227]. This study was supported by previous findings that riluzole reduces methamphetamine-induced locomotor sensitization [228]. Additionally, riluzole affects morphine- and amphetamine-conditioned place preference [229]. Riluzole also reduced ethanol self-administration in mice [230]. N-acetylcysteine (NAC), an antioxidant cystine pro-drug, is an over-the-counter supplement that suppresses NF-κB [116], a transcriptional regulator of EAAT2 [117]. While NAC has low oral availability, NAC has been shown to restore EAAT2 expression and to reduce drug-seeking/taking behavior for cocaine [118], nicotine [119], and ethanol [120].

Another promising approach for targeting EAAT2 in the context of addiction is by increasing the efficiency of the transport through PAMs. EAAT2 PAMs offer the unique advantage of having a quick onset, which may be of critical importance during craving.

3.9 Neurodegenerative disorders

Alzheimer’s disease (AD) is a progressive age-related neurodegenerative disorder characterized by abnormal deposition of fibrillar amyloid β (Aβ) protein, intracellular neurofibrillary tangles, oxidative damage, and tau protein hyperphosphorylation that contributes to neuronal dysfunction [231, 232, 233]. The aberrant glutamate stimulation that results in synaptic dysfunction has been proposed as one of several mechanisms of synaptic damage in AD. This is supported by studies reporting reduced GLT-1 expression and function in AD [234, 235] and post-mortem analysis of human brain tissue from patients with AD (as well as other neurodegenerative disorders), suggesting that EAAT2 loss or dysfunction could be an early trigger evolving into chronic reactive astrogliosis, oxidative stress, and neuronal death [236]. Also, a study demonstrated that Aβ1–42 prompts rapid GLT-1 mislocalization and internalization in astrocytes, reducing the rate of glutamate clearance [237]. Another study found that GLT-1 loss led to a compensatory increase in insulin-degrading enzyme activity in the liver, implicating partial GLT-1 loss in insulin/Akt signaling abnormalities observed in AD [238]. Further, a study suggested that decreased expression of glutamine synthetase but no changes in GLT-1 expression in a model of AD [239]. Thus, it is not yet entirely clear whether GLT-1/EAAT2 dysfunction plays a pathogenic role in AD and whether targeting this transporter may be developed as a neuroprotective strategy for the pathogenesis of AD.

Currently, available treatments for AD include acetylcholinesterase inhibitors [240], NMDA receptor antagonist memantine [241], and monoclonal antibody therapies that target the removal of β-amyloid from the brain such as lecanemab [242]. However, these treatments offer only symptomatic relief and primarily target late-stage aspects of the disease. Additionally, the efficacy of monoclonal antibodies has not been proven. Therefore, a definite treatment for this disease is yet to be identified.

Amyotrophic lateral sclerosis (ALS): ALS is a debilitating disease characterized by progressive loss of voluntary motor neurons, leading to muscle atrophy, weight loss, and respiratory failure [243]. The pathogenesis of ALS involves inflammation, oxidative stress, apoptosis, dysfunction of mitochondria, aggregation of SOD1 protein, and dysfunction of astroglia, which include a severe loss of EAAT2 in both the motor cortex and spinal cord [244, 245]. Additionally, several mouse models of ALS exhibit marked loss or inactivation of glutamate transporters [246]. Moreover, selective loss of EAAT2 has also been demonstrated in both sporadic and familial cases of ALS [247]. However, a large-scale clinical trial testing the efficacy of ceftriaxone (an EAAT2 expression enhancer) in ALS patients reported no significant difference in survival between placebo- and ceftriaxone-treated patients [248].

While glutamate transport dysfunction and excitotoxicity may contribute to the late stage of ALS disease progression, there is no consistent evidence supporting EAAT2 as a primary factor in motor neuron degeneration in ALS. A study found that overexpression of GLT-1 in the cervical spinal cord of SOD1G93A mice with ALS did not protect motor neurons, preserve diaphragm function, or prolong animal survival, thus challenging the notion that EAAT2 is a primary factor in motor neuron degeneration in ALS [249]. It is thought that simultaneous targeting of calcium overload, endoplasmic reticulum stress, and mitochondrial dysfunction pathways may be necessary to halt ALS progression [250].

Huntington’s disease (HD): HD is a devastating neurodegenerative disorder characterized by degeneration of multiple brain areas, involving dopamine, glutamate, and GABA neurotransmitter systems. It is caused by a mutated form of the huntingtin gene, resulting in the accumulation of mutant protein aggregates [251], resulting in oxidative stress, mitochondrial dysfunction, and excitotoxicity [252]. Reduced EAAT2 mRNA levels have been observed in HD patients [253], and transgenic mouse models, while increased GLT-1 expression can improve behavioral symptoms in HD mouse models [254]. Additionally, EAAT3 expression has been shown to be reduced in HD; however, this appears to be associated with issues with cysteine transport and oxidative stress rather than increases in extracellular glutamate concentration.

These studies suggest that alterations in EAAT2 and EAAT3 expression or function can influence the progression of Huntington’s disease (HD). However, effective therapies for HD have yet to be discovered and developed [71, 255, 256].

Human idiopathic Parkinson’s disease (PD): is a progressive neurodegenerative movement disorder characterized by the degeneration of dopaminergic neurons, leading to increased firing rates of glutamatergic excitatory projections to the substantia nigra [257]. This disturbance may contribute to glutamate-mediated excitotoxicity, exacerbating nigrostriatal degeneration in PD. Studies using unilateral 6-hydroxydopamine (6-OHDA) animal models of PD have demonstrated a link between disturbed glutamatergic neurotransmission and glutamate transporter functioning in the striatum. A study revealed posttranslational modifications on GLT-1, resulting in transporter trafficking by Nedd4-2 in a PD model. Additionally, rapamycin protects mice against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced loss of dopaminergic neurons in a PD model, through preservation of EAAT2, an effect mediated by NF-κB. Moreover, it was reported that increased expression of GLT-1 with ceftriaxone ameliorated locomotor impairments in a PD model [258].

Dyskinesias are a motor complication that develops as common side effect of current PD treatments such as L-DOPA (L-3,4-dihydroxyphenylalanine), which is used to replenish dopamine levels. Dyskinesias are linked to elevated extracellular glutamate levels in the basal ganglia, thus targeting EAAT2 modulation may represent a potential therapeutic target to treat them.

Collectively, it seems that dysregulation or loss of EAATs function, especially, EAAT2, can lead to glutamatergic excitotoxicity and neuronal death, contributing to neurodegenerative diseases such as AD, ALS, HD, and PD. However, our current understanding of the contribution of EAATs in these diseases is primarily based on experimental models and post-mortem brain tissue analysis. Detecting EAAT2 in living human brains could greatly improve diagnosis and therapy for these neurodegenerative disorders, and this could allow the possibility of utilizing EAAT2 activation for therapeutic interventions.

3.10 Other disorders

HIV-associated neurocognitive disorder (HAND): A common neuropathology observed in the brains of HIV-infected individuals is the excess release of glutamate upon HIV infection of macrophage/microglial cells [71, 259]. This has been linked to neurotoxicity mediated by various HIV proteins, including gp120 and transactivator of transcription (TAT). NMDA receptor antagonists were not effective at mitigating glutamate excitotoxicity in HAND due to side effects. Therefore, alternative approaches are being pursued, such as modulating the activity or expression of glutamate transporters. A study found that methamphetamine and HIV treatment activate trace amine-associated receptor 1 (TAAR1) in human astrocytes, leading to reduced EAAT2 mRNA levels and impaired glutamate clearance. CCL2 impairs spatial memory and cognition, potentially through upregulating mRNA expression linked to inflammation, excitotoxicity, and neuronal apoptosis in HAND. However, our understanding of the host factors contributing to the neurotoxic effects of HIV-1 on the CNS is evolving, and identification of strategies to mitigate the neurotoxic effects of viral and host proteins is crucial to develop neuroprotection strategies to alleviate the detrimental impact of HIV-1 on the brain.

Autism: The etiology of autism spectrum disorder is complex and involves genetic predisposition, environmental influences, and other yet unknown factors. It is thought that glutamate excitotoxicity, mitochondrial dysfunction, and degeneration are key components of autism. A report by the Autism Genome Project Consortium identified a linkage peak for autism in the region of chromosome 11, where the gene for EAAT2 is situated [260]. There is evidence suggesting astroglial dysfunction in the autistic brain and activators of GLT-1 expression have been shown to ameliorate certain symptoms of autism and reduce epilepsy seizures, suggesting that GLT-1 may be a novel therapeutic strategy for autism.

Additionally, EAAT2 dysfunction has been implicated in the pathogenesis of major depressive disorders [261], mood disorders [262, 263], glioma [264, 265], multiple sclerosis [266], and schizophrenia [267], among others. Dysfunction of EAAT3 has also been observed in schizophrenia [13] and OCD [268].

Collectively, these studies suggest that therapies aimed at the modulation of the function and/or expression of EAATs, particularly EAAT2, could have broad therapeutic potential for various CNS disorders.

4. Conclusions

EAATs are key proteins that regulate the excitatory tone in CNS and are important for many physiological functions. Dysfunction in their activity or expression has profound effects that have been implicated in the etiology of many acute and chronic pathologies. In the past decade, it has become evident that drugs targeting NMDA receptors and secondary damages from glutamate-mediated excitotoxicity are limited and ineffective, often resulting in unwanted side effects; thus, there is a need for better therapeutics. In this regard, enhancing glutamate transporter expression or function pharmacologically holds great promise for therapeutic interventions, particularly targeting EAAT2 [71, 269].

Drug development targeting EAATs started with the discovery of pharmacological agents that were developed to study the intrinsic properties and function of the EAATs, specifically the potent EAAT2 inhibitors TBOA and analogs [68, 71]. TBOA was the first non-transportable blocker for all subtypes of EAATs identified, which helped elucidate several functions of the EAATs, and encouraged the search for other modulators of the function of EAATs. However, there is also a need to identify subtype-selective enhancers and inhibitors for all subtypes of glutamate transporters, to fully understand how to fine-tune the extracellular concentration of glutamate in the CNS. Several EAAT2 PAMs have already been identified, which can be administered acutely and thus are advantageous over the expression enhancers that generally require prophylactic administration. It is yet to be established whether small molecule allosteric activators of EAAT2, like many biologics, can undergo traditional pharmacokinetic analysis and demonstrate efficacy in animal models of CNS disease. The future may uncover whether this class of compounds is effective in chronic conditions and when used in combination therapies. Moreover, the recent publication of cryo-EM structures of EAAT2 presents an opportunity to launch a structural-based drug design initiative aimed at screening and developing high-affinity EAAT2 PAMs. These compounds can have therapeutic potential and serve as imaging tools to detect changes in EAAT2 density in neurodegenerative diseases. Furthermore, the optimal timing for therapeutic intervention with EAAT2-targeting drugs remains uncertain. Hence, discovering noninvasive methods to enhance our understanding of EAAT2 function and expression in the living brain is imperative. Finally, there is still more to understand about the molecular behavior of glutamate transporters in dysfunctions. Current understanding suggests that a complex process is involved in the downregulation, reversal, and post-translational modifications of EAATs. The intracellular signaling pathways that accompany the changes in EAATs on disease states also need to be further understood.

In conclusion, EAAT dysfunction plays a significant role in many neuropsychiatric disorders. Recent advancements have propelled the pursuit of targeting these transporters as a strategy to prevent and treat glutamatergic dysfunctions. However, the full potential of modulating these transporters as a therapeutic target is yet to be fully understood, appreciated, and explored. We expect to see, in the upcoming years, the identification of an arsenal of selective pharmacological compounds that target these fascinating proteins and the investigation of their translational possibilities [71, 210].

Acknowledgments

ACKF received funding from NIH (RO1 NS111767), and KLR received financial support from the PhRMA foundation.

Conflict of interest

The authors declare no conflict of interest.