Solute carrier family 6 member 1 (SLC6A1) is abundantly expressed in the developing brain even before the CNS is formed. Its encoded GABA transporter 1 (GAT-1) is responsible for the reuptake of GABA into presynaptic neurons and glia, thereby modulating neurotransmission. GAT-1 is expressed globally in the brain, in both astrocytes and neurons. The GABA uptake function of GAT-1 in neurons cannot be compensated for by other GABA transporters, while the function in glia can be partially replaced by GABA transporter 3. Recently, many variants in SLC6A1 have been associated with a spectrum of epilepsy syndromes and neurodevelopmental disorders, including myoclonic atonic epilepsy, childhood absence epilepsy, autism, and intellectual disability, but the pathomechanisms associated with these phenotypes remain unclear. The presence of GAT-1 in both neurons and astrocytes further obscures the role of abnormal GAT-1 in the heterogeneous disease phenotype manifestations. Here we examine the impact on transporter trafficking and function of 22 SLC6A1 variants identified in patients with a broad spectrum of phenotypes. We also evaluate changes in protein expression and subcellular localization of the variant GAT-1 in various cell types, including neurons and astrocytes derived from human patient induced pluripotent stem cells. We found that a partial or complete loss-of-function represents a common disease mechanism, although the extent of GABA uptake reduction is variable. The reduced GABA uptake appears to be due to reduced cell surface expression of the variant transporter caused by variant protein misfolding, endoplasmic reticulum retention, and subsequent degradation. Although the extent of reduction of the total protein, surface protein, and the GABA uptake level of the variant transporters is variable, the loss of GABA uptake function and endoplasmic reticulum retention is consistent across induced pluripotent stem cell-derived cell types, including astrocytes and neurons, for the surveyed variants. Interestingly, we did not find a clear correlation of GABA uptake function and the disease phenotypes, such as myoclonic atonic epilepsy versus developmental delay, in this study. Together, our study suggests that impaired transporter protein trafficking and surface expression are the major disease-associated mechanisms associated with pathogenic SLC6A1 variants. Our results resemble findings from pathogenic variants in other genes affecting the GABA pathway, such as GABA_A receptors. This study provides critical insight into therapeutic developments for SLC6A1 variant-mediated disorders and implicates that boosting transporter function by either genetic or pharmacological approaches would be beneficial.

We have studied the molecular mechanisms of variants in solute carrier Family 6 Member 1 associated with neurodevelopmental disorders, including various epilepsy syndromes, autism and intellectual disability. Based on functional assays of solute carrier Family 6 Member 1 variants, we conclude that partial or complete loss of γ-amino butyric acid uptake due to reduced membrane γ-amino butyric acid transporter 1 trafficking is the primary aetiology. Importantly, we identified common patterns of the mutant γ-amino butyric acid transporter 1 protein trafficking from biogenesis, oligomerization, glycosylation and translocation to the cell membrane across variants in different cell types such as astrocytes and neurons. We hypothesize that therapeutic approaches to facilitate membrane trafficking would increase γ-amino butyric acid transporter 1 protein membrane expression and function. 4-Phenylbutyrate is a Food and Drug Administration-approved drug for paediatric use and is orally bioavailable. 4-Phenylbutyrate shows promise in the treatment of cystic fibrosis. The common cellular mechanisms shared by the mutant γ-amino butyric acid transporter 1 and cystic fibrosis transmembrane conductance regulator led us to hypothesize that 4-phenylbutyrate could be a potential treatment option for solute carrier Family 6 Member 1 mutations. We examined the impact of 4-phenylbutyrate across a library of variants in cell and knockin mouse models. Because γ-amino butyric acid transporter 1 is expressed in both neurons and astrocytes, and γ-amino butyric acid transporter 1 deficiency in astrocytes has been hypothesized to underlie seizure generation, we tested the effect of 4-phenylbutyrate in both neurons and astrocytes with a focus on astrocytes. We demonstrated existence of the mutant γ-amino butyric acid transporter 1 retaining wildtype γ-amino butyric acid transporter 1, suggesting the mutant protein causes aberrant protein oligomerization and trafficking. 4-Phenylbutyrate increased γ-amino butyric acid uptake in both mouse and human astrocytes and neurons bearing the variants. Importantly, 4-phenylbutyrate alone increased γ-amino butyric acid transporter 1 expression and suppressed spike wave discharges in heterozygous knockin mice. Although the mechanisms of action for 4-phenylbutyrate are still unclear, with multiple possibly being involved, it is likely that 4-phenylbutyrate can facilitate the forward trafficking of the wildtype γ-amino butyric acid transporter 1 regardless of rescuing the mutant γ-amino butyric acid transporter 1, thus increasing γ-amino butyric acid uptake. All patients with solute carrier Family 6 Member 1 variants are heterozygous and carry one wildtype allele, suggesting a great opportunity for treatment development leveraging wildtype protein trafficking. The study opens a novel avenue of treatment development for genetic epilepsy via drug repurposing.

The epilepsies are a broad group of conditions characterized by repeated seizures, and together are one of the most common neurological disorders. Additionally, epilepsy is comorbid with many neurological disorders, including lysosomal storage diseases, syndromic intellectual disability, and autism spectrum disorder. Despite the prevalence, treatments are still unsatisfactory: approximately 30% of epileptic patients do not adequately respond to existing therapeutics, which primarily target ion channels. Therefore, new therapeutic approaches are needed. Disturbed proteostasis is an emerging mechanism in epilepsy, with profound effects on neuronal health and function. Proteostasis, the dynamic balance of protein synthesis and degradation, can be directly disrupted by epilepsy-associated mutations in various components of the ubiquitin-proteasome system (UPS), or impairments can be secondary to seizure activity or misfolded proteins. Endoplasmic reticulum (ER) stress can arise from failed proteostasis and result in neuronal death. In light of this, several treatment modalities that modify components of proteostasis have shown promise in the management of neurological disorders. These include chemical chaperones to assist proper folding of proteins, inhibitors of overly active protein degradation, and enhancers of endogenous proteolytic pathways, such as the UPS. This review summarizes recent work on the pathomechanisms of abnormal protein folding and degradation in epilepsy, as well as treatment developments targeting this area.

Lennox–Gastaut Syndrome (LGS) is a developmental and epileptic encephalopathy (DEE) characterized by multiple seizure types, electroencephalogram (EEG) patterns, and cognitive decline. Its etiology has a prominent genetic component, including variants in GABRB3 that encodes the GABA_A receptor (GABA_AR) β₃ subunit. LGS has an unknown pathophysiology, and few animal models are available for studying LGS. The objective of this study was to evaluate Gabrb3^+/N328D knock-in mice as a model for LGS. We generated a heterozygous knock-in mouse expressing Gabrb3 (c.A982G, p.N238D), a de novo mutation identified in a patient with LGS. We investigated Gabrb3^+/N328D mice for features of LGS. In 2–4-month-old male and female C57BL/J6 wild-type and Gabrb3^+/N328D mice, we investigated seizure severity using video-monitored EEG, cognitive impairment using a suite of behavioral tests, and profiled GABA_AR subunit expression by Western blot. Gabrb3^+/N328D mice showed spontaneous seizures and signs of cognitive impairment, including deficits in spatial learning, memory, and locomotion. Moreover, Gabrb3^+/N328D mice showed reduced β₃ subunit expression in the cerebellum, hippocampus, and thalamus. This phenotype of epilepsy and neurological impairment resembles the LGS patient phenotype. We conclude that Gabrb3^+/N328D mice provide a good model for investigating the pathophysiology and therapeutic intervention of LGS and DEEs.

We have previously characterized the molecular mechanisms for variants in γ-aminobutyric acid transporter 1-encoding solute carrier family 6-member 1 (SLC6A1) in vitro and concluded that a partial or complete loss of γ-aminobutyric acid uptake due to impaired protein trafficking is the primary aetiology. Impairment of γ-aminobutyric acid transporter 1 function could cause compensatory changes in the expression of γ-aminobutyric acid receptors, which, in turn, modify disease pathophysiology and phenotype. Here we used different approaches including radioactive ³H γ-aminobutyric acid uptake in cells and synaptosomes, immunohistochemistry and confocal microscopy as well as brain slice surface protein biotinylation to characterize Slc6a1^+/A288V and Slc6a1^+/S295L mice, representative of a partial or a complete loss of function of SLC6A1 mutations, respectively. We employed the γ-aminobutyric acid transporter 1-specific inhibitor [³H]tiagabine binding and GABA_A receptor subunit-specific radioligand binding to profile the γ-aminobutyric acid transporter 1 and GABA_A receptor expression in major brain regions such as cortex, cerebellum, hippocampus and thalamus. We also determined the total and surface expression of γ-aminobutyric acid transporter 1, γ-aminobutyric acid transporter 3 and expression of GABA_A receptor in the major brain regions in the knockin mice. We found that γ-aminobutyric acid transporter 1 protein was markedly reduced in cortex, hippocampus, thalamus and cerebellum in both mutant mouse lines. Consistent with the findings of reduced γ-aminobutyric acid uptake for both γ-aminobutyric acid transporter 1(A288V) and γ-aminobutyric acid transporter 1(S295L), both the total and the γ-aminobutyric acid transporter 1-mediated ³H γ-aminobutyric acid reuptake was reduced. We found that γ-aminobutyric acid transporter 3 is only abundantly expressed in the thalamus and there was no compensatory increase of γ-aminobutyric acid transporter 3 in either of the mutant mouse lines. γ-Aminobutyric acid transporter 1 was reduced in both somatic regions and nonsomatic regions in both mouse models, in which a ring-like structure was identified only in the Slc6a1^+/A288V mouse, suggesting more γ-aminobutyric acid transporter 1 retention inside endoplasmic reticulum in the Slc6a1^+/A288V mouse. The [³H]tiagabine binding was similar in both mouse models despite the difference in γ-aminobutyric acid uptake function and γ-aminobutyric acid transporter 1 protein expression for both mutations. There were no differences in GABA_A receptor subtype expression, except for a small increase in the expression of α5 subunits of GABA_A receptor in the hippocampus of Slc6a1^S295L homozygous mice, suggesting a potential interaction between the expression of this GABA_A receptor subtype and the mutant γ-aminobutyric acid transporter 1. The study provides the first comprehensive characterization of the SLC6A1 mutations in vivo in two representative mouse models. Because both γ-aminobutyric acid transporter 1 and GABA_A receptors are targets for anti-seizure medications, the findings from this study can help guide tailored treatment options based on the expression and function of γ-aminobutyric acid transporter 1 and GABA_A receptor in SLC6A1 mutation-mediated neurodevelopmental and epileptic encephalopathies.

A significant number of patients with genetic epilepsy do not obtain seizure freedom, despite developments in new antiseizure drugs, suggesting a need for novel therapeutic approaches. Many genetic epilepsies are associated with misfolded mutant proteins, including GABRG2(Q390X)-associated Dravet syndrome, which we have previously shown to result in intracellular accumulation of mutant GABA_A receptor γ2(Q390X) subunit protein. Thus, a potentially promising therapeutic approach is modulation of proteostasis, such as increasing endoplasmic reticulum (ER)-associated degradation (ERAD). To that end, we have here identified an ERAD-associated E3 ubiquitin ligase, HRD1, among other ubiquitin ligases, as a strong modulator of wildtype and mutant γ2 subunit expression. Overexpressing HRD1 dose-dependently reduced the γ2(Q390X) subunit. Additionally, we show that zonisamide (ZNS)—an antiseizure drug reported to upregulate HRD1—reduces seizures in the Gabrg2^+/Q390X mouse. We propose that a possible mechanism for this effect is a partial rescue of surface trafficking of GABA_A receptors, which are otherwise sequestered in the ER due to the dominant-negative effect of the γ2(Q390X) subunit. Furthermore, this partial rescue was not due to changes in ER chaperones BiP and calnexin, as total expression of these chaperones was unchanged in γ2(Q390X) models. Our results here suggest that leveraging the endogenous ERAD pathway may present a potential method to degrade neurotoxic mutant proteins like the γ2(Q390X) subunit. We also demonstrate a pharmacological means of regulating proteostasis, as ZNS alters protein trafficking, providing further support for the use of proteostasis regulators for the treatment of genetic epilepsies.