Functional Characterization and Rescue of GABA Uptake in Human Ipsc-derived Gabaergic Neurons Carrying SLC6A1 Patient Mutations
Abstract number :
3.202
Submission category :
2. Translational Research / 2B. Devices, Technologies, Stem Cells
Year :
2024
Submission ID :
218
Source :
www.aesnet.org
Presentation date :
12/9/2024 12:00:00 AM
Published date :
Authors :
Presenting Author: Martine Geraerts, PhD – UCB Biopharma
Laura Backes, MSN – UCB Biopharma
Melvin Rincon, PhD, MD – UCB Biopharma
Seon-Ah Chong, PhD – UCB Biopharma
Fhilippe Motte, MSN – UCB Biopharma
Catherine Vandenplas, MSN – UCB Biopharma
Isabelle Niespodziany, PhD – UCB Pharma
Christian Wolff, PhD – UCB Pharma
Brittany Vallette, PhD – UCB Biopharma
Michel Gillard, PhD – UCB Biopharma
Rationale: Gamma-aminobutyric acid transporter 1 (GAT-1) encoded by SLC6A1 gene is one of the main GABA transporters that maintains GABA homeostasis and contributes to synaptic inhibition in the mammalian brain. More than 100 pathogenic variants in SLC6A1 gene have been identified and implicated in various neurological disorders including childhood-onset epilepsy, neurodevelopmental delay and movement disorders1. Several efforts have been made to understand functional impact of the variants; however, most of the works were done in non-relevant human cell line (e.g., HEK293) overexpressing homozygous mutated forms therefore lacking phenotype-genotype correlation in the disease-relevant cell systems such as GABAergic neurons and astrocytes2,3. In this study, we aimed to evaluate the functional effects of SLC6A1 heterozygous mutations in human iPSCs differentiated into GABAergic neurons.
Methods: Human iPSCs with SLC6A1 heterozygous mutations (F270S+/- and A288V+/-) or homozygous KO (SLC6A1-/-) were generated by CRISPR technology. Genes encoding ASCL1 and DLX2 transcription factors were introduced to AAVS1 locus by flipase – ligase system to differentiate into GABAergic neurons. Characterization of the GABAergic neurons were performed by RT-qPCR and patch clamp between 14 and 28 days in vitro (DIV). [3H] GABA uptake was performed at DIV21 to assess GAT-1 function. GABAergic neurons at DIV12 were transduced with Lentiviral vectors (LVs) containing WT SLC6A1 gene and [3H] GABA uptake was performed at DIV21.
Results: The differentiation protocol using ASCL1 and DLX2 delivered efficient and fast maturation into GABAergic neurons from iPSCs within 3 weeks confirmed by the key GABAergic neuronal markers including GAD1, GAD2, VGAT1 and somatostatin. Spontaneous inhibitory post-synaptic currents with high frequency and amplitude were also observed at DIV24 indicating strong GABAergic inputs in these neurons. We found that GAT-1 function is completely abolished in SLC6A1-/- neurons when compared to WT. About 50% reduction of GAT-1 function was observed in neurons with F270S+/- and A288V+/- confirming haploinsufficiency of these pathogenic variants. Transduction with LVV-WT SLC6A1 significantly increased [3H] GABA uptake in F270S+/- and SLC6A1-/- neurons together with the increase in GAT-1 expression.
Conclusions: We confirmed significant reduction of GAT-1 function in human iPSC-derived GABAergic neurons harboring heterozygous SLC6A1 mutations. Viral vector delivery of WT SLC6A1 gene increased GAT-1 expression and function.
Funding: GREAT GRANT (Wallonie recherche SPW)
Translational Research