Genetic epilepsies refer to epilepsy syndromes previously classified as idiopathic generalized epilepsies and have been associated with gene mutations using current advances in genetics and sequencing technology.¹ There are many ion channel and non–ion channel gene mutations that have been identified in epilepsy.² These ion channel genes include those encoding proteins that form channels conducting sodium, potassium, calcium, and chloride ions. Those non–ion channel genes include an overwhelmingly comprehensive and still growing list of genes, with their function involved in protein endocytosis, synaptic development, and transcription, as well as many unknown mechanisms. To date, most functional studies of epilepsy genetic mutations have been focused on ion channel genes. Mutation of ion channels that cause either a “gain of function” in excitatory neurotransmission or a “loss of function” in inhibitory neurotransmission could impair the balance of excitation and inhibition, leading to disinhibition and hyperexcitability in the brain. In this review article, we focus on the molecular pathogenic basis for genetic generalized epilepsies associated with mutations in the inhibitory γ-aminobutyric acid (GABA_A) receptor γ2 subunit gene, GABRG2 (OMIM 137164), an established epilepsy gene that we have extensively characterized.

The Q390X mutation in the GABAA receptor GABRG2 has been associated with Dravet syndrome in humans. In this study, the authors generated a new genetic epileptic encephalopathy animal model, the Gabrg2+/Q390X knock-in mouse, and show that expression of this mutant protein leads to seizures, chronic accumulation and aggregation of mutant subunit protein and age-dependent neurodegeneration. Genetic epilepsy and neurodegenerative diseases are two common neurological disorders that are conventionally viewed as being unrelated. A subset of patients with severe genetic epilepsies who have impaired development and often go on to die of their disease respond poorly to anticonvulsant drug therapy, suggesting a need for new therapeutic targets. Previously, we reported that multiple GABAA receptor epilepsy mutations result in protein misfolding and abnormal receptor trafficking. We have now developed a model of a severe human genetic epileptic encephalopathy, the Gabrg2+/Q390X knock-in mouse. We found that, in addition to impairing inhibitory neurotransmission, mutant GABAA receptor γ2(Q390X) subunits accumulated and aggregated intracellularly, activated caspase 3 and caused widespread, age-dependent neurodegeneration. These findings suggest that the fundamental protein metabolism and cellular consequences of the epilepsy-associated mutant γ2(Q390X) ion channel subunit are not fundamentally different from those associated with neurodegeneration. Our results have far-reaching relevance for the identification of conserved pathological cascades and mechanism-based therapies that are shared between genetic epilepsies and neurodegenerative diseases.

Mutations in GABRB3, which encodes the β3 subunit of GABAA receptors, cause variable epilepsy syndromes with autism and intellectual disability. Shi et al. report that mutant β3 subunits reduce expression of wildtype γ2 subunits, which are critical for receptor synaptic clustering. However, they do so to different degrees, contributing to disease heterogeneity.

The GABA_A receptor γ2 subunit mutation, Q351X, associated with generalized epilepsy with febrile seizures plus (GEFS+), created a loss of function with homozygous expression. However, heterozygous γ2(+/−) gene deletion mice are seizure free, suggesting that the loss of one GABRG2 allele alone in heterozygous patients may not be sufficient to produce epilepsy. Here we show that the mutant γ2 subunit was immature and retained in the endoplasmic reticulum (ER). With heterozygous coexpression of γ2S/γ2S(Q351X) subunits and α1 and β2 subunits, the trafficking deficient mutant γ2 subunit reduced trafficking of wild-type partnering subunits, which was not seen in the hemizygous gene deletion control. Consequently, the function of the heterozygous receptor channel was reduced to less than the hemizygous control and to less than half of the wild-type receptors with a full gene dose. Pulse-chase experiments demonstrated that in the presence of the mutant γ2S(Q351X) subunit, wild-type α1 subunits degraded more substantially within 1 h of translation. We showed that the basis for this dominant-negative effect on wild-type receptors was due to an interaction between mutant and wild-type subunits. The mutant subunit oligomerized with wild-type subunits and trapped them in the ER, subjecting them to glycosylation arrest and ER-associated degradation (ERAD) through the ubiquitin proteosome system. Thus, we hypothesize that a likely explanation for the GEFS+ phenotype is a dominant-negative suppression of wild-type receptors by the mutant γ2S subunit in combination with loss of mutant γ2S subunit protein function.

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.

Approximately one-third of human genetic diseases are caused by premature translation-termination codon (PTC)-generating mutations. These mutations in sodium channel and GABA_Areceptor genes have been associated with idiopathic generalized epilepsies, but the cellular consequences of the PTCs on the mutant channel subunit biogenesis and function are unknown. The PTCs could result in translation of a truncated subunit, or more likely, trigger mRNA degradation through nonsense-mediated mRNA decay (NMD), thus preventing or reducing production of mutant subunit at the transcriptional level. The GABA_Areceptor α1 subunit mutation, 975delC, S326fs328X, is an autosomal dominant mutation associated with childhood absence epilepsy that generates a PTC in exon 8 of the 9 exon GABRA1 gene that is 74 bp upstream of intron 8. Using an intron 8-inclusion minigene that supports NMD, we demonstrated that mutant mRNA was substantially reduced, but not absent. Loss of mutant transcripts was blocked by ribosome inhibition or by silencing the NMD-essential gene hUPF-1. In both neurons and non-neuronal cells, the PTC caused substantial loss of mutant α1(S326fs328X) subunit mRNA through NMD with a minor portion of the mRNA escaping NMD and producing a mutant protein. The translated mutant protein had reduced stability due to endoplasmic reticulum associated degradation (ERAD) and had enhanced association with molecular chaperones. This study suggests that loss of mRNA due to activation of NMD and activation of ERAD by the mutant protein may contribute to epileptogenesis. The molecular mechanisms outlined here delineate a model for the pathogenesis of many PTC-generating mutations.