Human Ipsc Modeling of slc6a1-related Developmental and Epileptic Encephalopathy
Abstract number :
1.066
Submission category :
1. Basic Mechanisms / 1E. Models
Year :
2022
Submission ID :
2203924
Source :
www.aesnet.org
Presentation date :
12/3/2022 12:00:00 PM
Published date :
Nov 22, 2022, 05:22 AM
Authors :
Maria Varela, BA,BS – University of Michigan, Ann Arbor; Tyler Thenstedt, Undergradute Student – Undergraduate Research Assistant, Michigan Neuroscience Institute, Depatment of Neurology, University of Michigan; Michael Uhler, Ph.D. – Professor, Michigan Neuroscience Institute, Department of Biological Chemistry, Neuroscience Graduate Program, The University of Michigan, Ann Arbor; Jack Parent, M.D. – Professor, Michigan Neuroscience Institute, Depatment of Neurology, Neuroscience Graduate Program, The University of Michigan, Ann Arbor
This abstract has been invited to present during the Broadening Representation Inclusion and Diversity by Growing Equity (BRIDGE) poster session
Rationale: Myoclonic atonic epilepsy (MAE), a severe developmental and epileptic encephalopathy (DEE), is characterized by seizures, developmental delay, and intellectual disability. MAE, as well as other neurodevelopmental disorders, have been linked to mutations in the SLC6A1 gene encoding the most abundant brain GABA transporter, GAT-1. The GAT-1 transporter regulates the reuptake of GABA at the synapse and, in the cortex, is expressed primarily in interneurons. While loss of function (LOF) of GAT-1 leads to epilepsy and cognitive delays, understanding of how GAT-1 haploinsufficiency affects early human cortical development 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 interneuron development and subsequent network formation.
Methods: We reprogrammed human foreskin fibroblasts into iPSCs with concurrent CRISPR/Cas9 gene editing to generate out-of-frame insertions/deletions in the SLC6A1 gene. Compound heterozygous (KO), heterozygous (Het), and isogenic control (WT) iPSC lines were generated and differentiated into 3D ventral forebrain (VF)-like organoids using our novel self-organizing single rosette spheroid (SOSRS) model, or 2D induced GABAergic neurons (iGNs) using inducible expression of ASCL1 and DLX2. We performed immunolabeling and RT-qPCR of SOSRS cultured for 2-36 weeks or iGNs for 1-4 weeks to investigate interneuron development and specification. Cortical and VF fusion SOSRS were used to investigate interneuron migration.
Results: Both our 2D and 3D WT models displayed the expected GABAergic and mature neuronal marker expression and morphology. GAT-1 mRNA expression was decreased 80-95% in KO and 10-70% in HET iGNs and VF SOSRS compared to WT (n=3 lines each). We observed accelerated GABAergic neuron maturation in KO iGNs and VF SOSRS with earlier increases in potassium-chloride cotransporter 2 (KCC2) and somatostatin expression compared to WT. Additionally, a delayed migration phenotype was observed in mutant SLC6A1 VF SOSRS, with KO and Het SOSRS exhibiting reduced cell body migration (p= < 0.0001) and diminished process extension (p=0.002-0.03).
Basic Mechanisms