The severity of SLC1A2-associated neurodevelopmental disorders correlates with transporter dysfunction

L-Glutamate (L-Glu) is the major excitatory neurotransmitter in the human brain, where it is involved in or contributes to essentially all central functions. Glutamatergic hyper- or hypofunction have been linked to a range of neurological, cognitive, and psychiatric disorders, and thus glutamatergic receptors, transporters and enzymes constitute promising therapeutic targets.1, 2, 3Following its release from presynaptic terminals, L-Glu is quickly taken up into glia and neurons by five excitatory amino acid transporters (EAAT1-5) from solute carrier 1 (SLC1) family of Na+-dependent transporters.4, 5, 6 The EAAT functions as a homotrimeric assembly, with each monomer comprising intracellular amino- and carboxy-termini, eight transmembrane domains (TM1-8) and two hairpin structures (HP1-2) with interconnecting extra- and intracellular loops.5,7,8 The transport domain (TM3,6–8, HP1-2) in the monomer mediates stoichiometrically coupled L-Glu transport via an elevator mechanism driven by co-transport of three Na+ and one H+ and counter-transport of one K+, while the trimerization domain (TM1-2,4–5) is anchored to the plasma membrane making inter-monomeric contacts.5,7, 8, 9, 10 EAATs also act as anion channels that open and close during transitions along the L-Glu transport cycle via brief gap openings between the transport and trimerization domains.11, 12, 13, 14, 15Variants in SLC1A1-3 and SLC1A6-7 encoding for the five EAATs have been linked with various neurological and neuropsychiatric disorders.16,17 For example, we and others have found missense SLC1A3 variants associated with episodic ataxia 6 to change EAAT1 anion channel conduction.13,18, 19, 20, 21 EAAT2, the predominant glial and presynaptic EAAT subtype, is abundantly expressed throughout the brain, where it mediates ∼90% of total central L-Glu uptake,4,22,23 which makes SLC1A2 variants of potential importance for a range of central nervous system (CNS) disorders.24, 25, 26 Recently, three missense SLC1A2 variants associated with developmental and epileptic encephalopathies (DEEs)27, 28, 29, 30 were reported to significantly alter EAAT2 anion channel function.24 However, the SLC1A2-related phenotype remains ill-defined, and genotype-phenotype correlations have not been reported.With the present study we aim to provide the first genotype-phenotype correlation analysis for SLC1A2, to elucidate the molecular phenotypic basis for SLC1A2-associated neurodevelopmental disorders, and to provide the basis for future predictions of disease progression and severity of these. We present a cohort of 18 individuals with 13 presumed pathogenic SLC1A2 variants presenting with neurodevelopmental disorders and DEEs. We delineate the effects induced by nine novel missense variants on EAAT2 expression and function via heterologous expression of wild-type (WT) and mutant transporters in mammalian cell lines, and we deep-phenotype the neurodevelopmental disorders and epileptology associated with SLC1A2 variants.