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Infantile spasms are anis an epileptic encephalopathy of childhood, and its pathophysiology is largely unknown.We generated a heterozygous knock-in mouse with the human infantile spasms-associated de novo mutation GABRB3(c.A328G, p.N110D) to investigate its molecular mechanisms and to establish the Gabrb3We used electroencephalography (EEG) and video monitoring to characterize seizure types, and a suite of behavioral tests to identify neurological and behavioral impairment in Gabrb3The infantile spasms-associated human de novo mutation GABRB3(c.A328G, p.N110D) caused epileptic spasms early in development and multiple seizure types in adult Gabrb3The Gabrb3

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.

Mutations in SLC6A1, encoding γ-aminobutyric acid (GABA) transporter 1 (GAT-1), have been recently associated with a spectrum of epilepsy syndromes, intellectual disability and autism in clinic. However, the pathophysiology of the gene mutations is far from clear. Here we report a novel SLC6A1 missense mutation in a patient with epilepsy and autism spectrum disorder and characterized the molecular defects of the mutant GAT-1, from transporter protein trafficking to GABA uptake function in heterologous cells and neurons. The heterozygous missense mutation (c1081C to A (P361T)) in SLC6A1 was identified by exome sequencing. We have thoroughly characterized the molecular pathophysiology underlying the clinical phenotypes. We performed EEG recordings and autism diagnostic interview. The patient had neurodevelopmental delay, absence epilepsy, generalized epilepsy, and 2.5–3 Hz generalized spike and slow waves on EEG recordings. The impact of the mutation on GAT-1 function and trafficking was evaluated by 3H GABA uptake, structural simulation with machine learning tools, live cell confocal microscopy and protein expression in mouse neurons and nonneuronal cells. We demonstrated that the GAT-1(P361T) mutation destabilizes the global protein conformation and reduces total protein expression. The mutant transporter protein was localized intracellularly inside the endoplasmic reticulum (ER) with a pattern of expression very similar to the cells treated with tunicamycin, an ER stress inducer. Radioactive 3H-labeled GABA uptake assay indicated the mutation reduced the function of the mutant GAT-1(P361T), to a level that is similar to the cells treated with GAT-1 inhibitors. In summary, this mutation destabilizes the mutant transporter protein, which results in retention of the mutant protein inside cells and reduction of total transporter expression, likely via excessive endoplasmic reticulum associated degradation. This thus likely causes reduced functional transporter number on the cell surface, which then could cause the observed reduced GABA uptake function. Consequently, malfunctioning GABA signaling may cause altered neurodevelopment and neurotransmission, such as enhanced tonic inhibition and altered cell proliferation in vivo. The pathophysiology due to severely impaired GAT-1 function may give rise to a wide spectrum of neurodevelopmental phenotypes including autism and epilepsy.

Mutations in γ-aminobutyric acid (GABA) transporter 1 (GAT-1)-encoding SLC6A1 have been associated with myoclonic atonic epilepsy and other phenotypes. We determined the patho-mechanisms of the mutant GAT-1, in order to identify treatment targets.We conducted whole-exome sequencing of patients with myoclonic atonic epilepsy (MAE) and characterized the seizure phenotypes and EEG patterns. We studied the protein stability and structural changes with homology modeling and machine learning tools. We characterized the function and trafficking of the mutant GAT-1 withWe identified four novel SLC6A1 variants associated with MAE and 2 to 4 Hz spike-wave discharges as a common EEG feature. Machine learning tools predicted that the variant proteins are destabilized. The variant protein had reduced expression and reduced GABA uptake due to endoplasmic reticular retention. The consistent observation was made in cortical and thalamic astrocytes from variant-knockin mice and human iPSC-derived astrocytes. The Slc6aSLC6A1 variants in various locations of the protein peptides can cause MAE with similar seizure phenotypes and EEG features. Reduced GABA uptake is due to decreased functional GAT-1, which, in thalamic astrocytes, could result in increased extracellular GABA accumulation and enhanced tonic inhibition, leading to seizures and abnormal EEGs.

We have previously studied the molecular mechanisms of solute carrier family 6 member 1 (SLC6A1) associated with a continuum of neurodevelopmental disorders, including various epilepsy syndromes, autism, and intellectual disability. Based on functional assays of variants in a large cohort with heterogenous clinical phenotypes, we conclude that partial or complete loss of GABA uptake function in the mutant GAT-1 is the primary etiology as identified in GABAA receptor mutation-mediated epilepsy and in cystic fibrosis. Importantly, we identified that there are common patterns of the mutant protein trafficking from biogenesis, oligomerization, glycosylation, and translocation to the cell membrane across variants with the conservation of this process across cell types. Conversely any approach to facilitate membrane trafficking would increase presence of the functional protein in the targeted destination in all involved cells. PBA is an FDA-approved drug for pediatric use and is orally bioavailable so it can be quickly translated to patient use. It has been demonstrated that PBA can correct protein misfolding, reduce ER stress, and attenuate unfolded protein response in neurodegenerative diseases, it has also showed promise in treatment of cystic fibrosis. The common cellular mechanisms shared by the mutant GAT-1 and the mutant cystic fibrosis transmembrane conductance regulator led us to test if PBA and other pharmaco-chaperones could be a potential treatment option for SLC6A1 mutations. We examined the impact of PBA and other small molecules in a library of variants and in cell and knockin mouse models. Because of the critical role of astrocytic GAT-1 deficit in seizures, we focused on astrocytes, and demonstrated that the existence of the mutant GAT-1 retained the wildtype GAT-1, suggesting aberrant protein oligomerization and trafficking caused by the mutant GAT-1. PBA increased GABA uptake in both mouse and human astrocytes bearing the mutations. Importantly, PBA increased GAT-1 expression and suppressed spike wave discharges (SWDS) in the heterozygous knockin mice. Although the detailed mechanisms of action for PBA are ambiguous, it is likely that PBA can facilitate the forward trafficking of the wildtype GAT-1 favoring over the mutant GAT-1, thus increasing GABA uptake. Since all patients with SLC6A1 mutations are heterozygous and carry one wildtype functional allele, this suggests a great opportunity for treatment development by leveraging the endogenous protein trafficking pathway to promote forward trafficking of the wildtype in combination with enhancing the disposal of the mutant allele as treatment mode. The study opens a novel avenue of treatment development for genetic epilepsy via drug repurposing.