The Numerous Ways Glutamate Transporters can go Wrong

Abstract

The Severity of SLC1A2-Associated Neurodevelopmental Disorders Correlates With Transporter Dysfunction

Kovermann P, Bayat A, Fenger CD, Leeuwen L, Borovikov A, Sharkov A, Levrat V, Lesca G, Perrin L, Levy J, Fahlke C, Møller RS, Jensen AA. EBioMedicine. 2025;114:105648.

Background: Excitatory amino acid transporter 2 (EAAT2) is the predominant glutamate transporter and a key mediator of excitatory neurotransmission in the human brain. Here we present a cohort of 18 individuals harbouring 13 different SLC1A2 variants, who all present with neurodevelopmental impairment with variable symptoms and disease severities, and we delineate the impact of these variants on EAAT2 function. Methods: The consequences of 9 novel missense SLC1A2 variants for expression, transport, and anion channel properties of EAAT2 expressed in mammalian cells were characterized by confocal microscopy, enzyme-linked immunosorbent and [3H]-D-aspartate uptake assays, and electrophysiological recordings. Findings: Ten of the 13 SLC1A2 variants mediated significant changes to EAAT2 expression and/or function. These molecular phenotypes were classified into 3 categories: overall loss-of-function (F249Sfs∗17, A432D, A439V, c.1421+1G > C), mild gain-of-anion-channel function (I276S, G360A), and mixed loss-of-transport/gain-of-anion-channel function (G82R, L85R, L85P, P289R). In contrast, L37P, H542R, and I546T did not mediate significant changes to EAAT2 expression or function. Although specific clinical outcomes in individuals carrying variants within each category varied somewhat, the 3 categories overall translated into distinct clinical phenotypes in terms of phenotypic traits and severity. Interpretation: The observed associations between functional effects and clinical phenotypes produced by these variants offer valuable insights for future predictions of progression and severity of SLC1A2-associated neurodevelopmental disorders. Furthermore, these associations between variant-induced changes in EAAT2 function and phenotypic traits could assist in tailoring personalized treatments of these disorders.

Commentary

Early in the 1940s, the Japanese researcher Takashi Hayashi found that injection of glutamate at molar doses into the cortical gray matter of dogs (and several humans, too) can induce clonic convulsions. Hayashi suggested that epileptic seizures manifest when the “quantity of the glutamic acid in human brain surpasses the critical level.”^1,2 Three decades later, the mechanism for glutamate removal from the synaptic cleft was found to be sodium-dependent cellular uptake, later attributed to excitatory amino acid transporters (EAATs).^3,4

Among the 5 EAATs, excitatory amino acid transporter 2 (EAAT2) stands out as the one responsible for approximately 90% of glutamate reuptake. The EAAT2 protein forms trimeric functional complexes, which are then trafficked to the cell membrane. EAAT2 and other EAATs have a dual function, as glutamate/sodium/potassium/proton cotransporters, and as anion channels. ⁵ Given the complex processes involved in EAAT trafficking, gating, and transport, genetic variation largely implies that if anything can go wrong, it will. This is well demonstrated in the featured study, in which Kovermann and colleagues characterized in-depth 13 SLC1A2 variants (including 9 novel mutations) that lead to changes at various locations of the EAAT2 protein. ⁶ The variants were identified in 18 individuals with neurodevelopmental disorders, including 7 previously reported individuals. Carriers of pathogenic or likely pathogenic variants were 20 months of age to 44 years old (5 females and 10 males). The variants A439V and c.1421+1G >C were present in a homozygous state in 1 individual each.

Three mutations encoded proteins that were not inserted into the cell membrane, and 1 (I276S) resulted in an EAAT2 variant whose expression tended to be lower than that of the wild-type transporter. The cell surface expression of 3 other mutations was elevated. Because in most carriers the variants were heterozygous, mutant alleles that impaired the insertion of the protein into membranes were additionally coexpressed with wild-type SLC1A2, resulting in poor or modest cell surface transporter abundance. The expression levels overall correlated with the uptake capacity of radiolabeled aspartate, except for the aforementioned A439 V variant that was well expressed, but the protein exhibited compromised transport activity. Measurement of anion currents identified variants causing robust gain-of-function, mild gain-of-function, loss-of-function, and those without measurable effects on anion channel function.

Based on these phenotypes, the variants mediating significant changes in EAAT2 expression and/or function were classified into 3 categories: (1) loss-of-function: the carriers exhibited intermediate phenotype severity and heterogeneity in specific symptoms (normal intelligence to more pronounced cognitive impairment, with or without vision loss, and all 4 individuals had epilepsy). Within this group, the variant A439 V was pathogenic only in its homozygous state. The other individual who was homozygous for a variant SLC1A2 allele (c.1421+1G >C) exhibited relatively mild symptoms. (2) Mild gain-of-anion-channel function: These variants, including I276S, were categorized as those associated with little change in glutamate transport; hence, the clinical symptoms were attributed to excessive channel activity. Although the modestly augmented anion conductance led to the mildest clinical phenotypes, it was still associated with severe intellectual disability. (3) Mixed loss-of-transport/gain-of-anion-channel function: Phenotypes of this group were the most severe, including developmental and epileptic encephalopathies or infantile epileptic spasm syndrome and profound intellectual disability. The gain-of-channel function component was assumed to play a major role in the clinical manifestations, which were more severe than those caused by the loss-of-function mutations. As to epilepsy, the authors speculated that it was associated with reduced glutamate transport by EAAT2: first, epilepsy was present regardless of the ion channel status; and second, whereas among the 15 carriers of pathogenic variants, the only 2 individuals who did not have epilepsy carried the only variant with near-normal glutamate transport capacity (G360A).

The establishment of genotype–phenotype relationships is remarkable, given the multifaceted nature of EAAT2. Many mysteries have yet to be resolved, though. For example, homozygous Slc1a2^−/− mice exhibited severe epilepsy with poor survival at 6 weeks, ⁷ and eaat2a^−/− zebrafish mutants were not viable past larval stage. ⁸ Luckily, the human phenotype was milder: the homozygous carrier of c.1421+1G >C (that eliminates EAAT2 expression) was 2 years old at seizure onset, and his intellectual and social-emotional development normalized by 12 years. Whether the difference in severity is related to compensatory mechanisms in humans, polygenic effects, or other factors, this phenomenon requires further attention. The homozygous carriers raise several additional questions, for example, about the prevalence of such variants, which might direct genetic counseling.

Several study limitations are inherent to the rarity of the mutations and the nature of patient recruitment. For example, the small number of mutations and individuals does not allow for estimating the consistency of the genotype–phenotype relationships. Another limitation is associated with the use of “historic controls.” Indirect comparisons of membrane expression, transport activity, and electrophysiological function with those of the previously published variants might slightly confound future modeling of EAAT2 activity.

EAAT2 function is controlled not only by intrinsic but also by extrinsic factors. One such regulator is cholesterol 24-hydroxylase (CH24H), which is induced in astrocytes in temporal lobe epilepsy. Overactive CH24H may lead to loss of EAAT2 association with the lipid rafts and to reduced glutamate uptake function (similar to several loss-of-function mutations). The experimental compound soticlestat (TAK-935) has been suggested to preserve the membrane raft structure by inhibiting the CH24H-mediated catabolism of membrane cholesterol in the rafts. ⁹ Theoretically, it could improve the outcomes in carriers of variants that lead to partial transporter expression on cell membranes. However, the soticlestat development program was discontinued in January 2025 after missing the primary endpoints in 2 phase 3 trials. A novel selective positive allosteric EAAT2 modulator, iQ-007, will soon be evaluated in a phase 1 trial (NCT06899230). It would be interesting to assess the effects of this compound on the functional activity of variant transporters.

The study by Kovemann et al demonstrates the challenges and opportunities in investigating EAAT mutations. It highlights that extracellular accumulation of glutamate is not the only outcome of EAAT2 mutations, nor is it necessarily the worst outcome—even considering the harmful effects of excessive synaptic glutamate and its potential association with epilepsy. Further research into the regulation of EAAT2 function, including how loss-of-function mutations are compensated for in humans, might lead to the development of new treatments for carriers of these variants.