Rationale:
Developmental and epileptic encephalopathies (DEEs) are characterized by severe seizures, developmental delays, and intellectual disabilities. Recent studies implicate SLC6A1 loss of function (LOF) in myoclonic-atonic epilepsies and other DEEs. SLC6A1 encodes for the GABA transporter 1 protein (GAT1), which is expressed primarily on GABAergic neurons in the cortex, and is responsible for the reuptake of GABA at the synapse. The GABAergic system has been shown to play an important role in cortical development and its dysfunction has been linked to epilepsy, but how SLC6A1 LOF affects the early development and maturation of cortical neurons remains unknown. We hypothesize that increases in ambient GABA due to SLC6A1 LOF will result in the altered development of human cortical neurons.
Methods:
CRISPR/CAS9 gene edited SLC6A1 knockout (KO) and isogenic control (WT) human IPSC lines were used to generate self-organizing single-rosette cortical organoids (SOSR-COs). Due to limited access to human fetal tissue, a physiologically relevant human-derived model is essential given the limitations of mouse models in recapitulating human-specific phenotypes of SLC6A1 DEEs. Organoids were collected at 1-, 2-, 3-, and 5-months in vitro, and immunostained, confocal imaged, and analyzed for expression of markers related to human cortical development.
Results:
We found that HOPX+ outer radial glial cells are present in both KO and isogenic control SOSR-COs. HOPX+ cells in KO organoids, however, were localized more on the periphery compared to WT. Additionally, the expression of TBR2+ intermediate progenitor cells appeared earlier and in greater numbers in the KO organoids compared to WT from 2-5 months in culture. The number of cortical layer-specific putative excitatory neurons was also increased in the KO vs WT including upper, mid, and deep layer neurons expressing CUX1, SATB2, CTIP2, respectively.
Conclusions:
These findings suggest that the observed increased expression of intermediate progenitors results in an overproduction of cortical neurons that spans across multiple cortical layers. This indicates that SLC6A1 LOF alters cortical neuron development and affects the formation and organization of neurons in the cortex. Alterations in cortical neuron maturation timing and localization could lead to dysregulated network formation and ultimately result in epilepsy. Further investigation could offer additional mechanistic insight into the pathogenesis of SLC6A1-related DEEs and lead to potential treatment targets for these patients. Funding:
This work was supported by the AES 2025 Summer BRIDGE Internship Grant, NIH (NINDS) U54NS117170 (JMP), the NSF Graduate Research Fellowship Program (MCV) and the NIH (NINDS) DSPAN F99/K00 predoc to postdoc transition award(MCV).