Abstracts

Rationale: Myoclonic atonic epilepsy (MAE), a severe developmental and epileptic encephalopathy (DEE), is characterized by severe seizures, developmental delays, and intellectual disability. MAE is linked to mutations in the SLC6A1 gene which encodes for the most common GABA transporter in the brain, GAT-1. GAT-1 is responsible for the reuptake of GABA at the synapse and, in the cortex, is expressed primarily in inhibitory neurons. While loss-of-function (LOF) of GAT-1 leads to epilepsy and cognitive delays, a solid characterization of how GAT-1 haploinsufficiency affects early cortical development in humans remains elusive. Here, we use both 2D and 3D human induced pluripotent stem cell (iPSC)-derived models to test the hypothesis that GAT-1 LOF alters inhibitory neuron development and subsequent network formation.

Methods: We first reprogrammed human foreskin fibroblasts into iPSCs with concurrent CRISPR/Cas9 gene editing targeted to generate out-of-frame indels in the SLC6A1 gene. Heterozygous (Het), compound heterozygous (KO), and isogenic control (WT) iPSC lines were generated and differentiated into 3D MGE-like organoids using our novel self-organizing single rosette spheroid (SOSRS) model, or 2D induced GABAergic neurons (iGNs) using an inducible expression system driving human ASCL1 and DLX2. Organoid fusion protocols were also used to fuse cortical SOSRS with MGE-like SOSRS to investigate interneuron migration and integration into the cortex. Immunostaining and qRT-PCR were used to evaluate relevant marker expression.

Results: Both our 2D and brain organoid models displayed the expected GABAergic and mature neuronal marker expression and morphology. We found a greater than 50% reduction of GAT-1 expression in the KO iGNs and SOSRS and varying degrees of reduction in the Het lines compared to WT. We observed a potential delayed migration phenotype in mutant SLC6A1 SOSRS compared to controls, with the KO SOSRS displaying severe delays in neuronal migration, and Hets showing an intermediate degree of decreased migration.

Conclusions: Ambient GABA levels have been shown to affect interneuron migration in development. Our findings suggest that loss-of-function mutations in the SLC6A1 gene that are predicted to increase ambient GABA levels in early brain development would have deleterious effects on GABAergic cell migration and subsequent network formation. Further investigation into this phenotype could provide early treatment targets for SLC6A1-related disorders.

Funding: Please list any funding that was received in support of this abstract.: This work was supported by NIH (NINDS) U54NS117170 (JMP) and the NSF Graduate Research Fellowship Program (MCV).