Authors :
Presenting Author: Leah Goldstein, Undergraduate – University of Michigan. Ann Arbor
M. Carmen Varela, BSc Ba – University of Michigan
Tyler Thenstedt, BS – University of Michigan. Ann Arbor
Michael Uhler, PhD – University of Michigan. Ann Arbor
Jack Parent, MD – University of Michigan
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 loss-of-function (LOF) variants in the SLC6A1 gene encoding the most abundant brain GABA transporter, GAT-1. GAT-1 regulates the synaptic reuptake of GABA and, in the cortex, is expressed primarily in interneurons. While LOF of GAT-1 leads to epilepsy and cognitive delays, how GAT-1 loss affects cortical interneuron development remains elusive. Here, we use a medial ganglionic eminence (MGE) brain organoid (MGEO) model generated from human induced pluripotent stem cells (iPSCs) to test the hypothesis that GAT-1 loss alters interneuron development.Methods:
Human fibroblasts were reprogrammed into iPSCs with concurrent CRISPR/Cas9 gene editing to target SLC6A1 knockout (KO). Compound heterozygous (KO), heterozygous (Het), and isogenic control (WT) iPSC lines were generated, as were MAE patient and sex-matched control iPSC lines . We performed bulk RNA sequencing of MGEOs at 21 days in vitro (DIV) and immunolabeling and RT-qPCR of 30-90 DIV MGEOs to investigate interneuron development and specification.
Results:
Reduction of GAT-1 mRNA expression was confirmed in SLC6A1 mutant MGEOs using qRT-PCR. GAT-1 expression decreased 80-95% in KO and 10-70% in HET lines vs. WT. We observed early upregulation of potassium-chloride cotransporter 2 (KCC2) and MAP2 in KO MGEOs vs. control, as well as a transient increases in SST expression in KO MGEOs beginning at 25 DIV, followed by a reduction at later stages, potentially indicating accelerated neuronal maturation and altered chloride homeostasis. Additionally, differential gene expression analysis of bulk RNA sequencing of SLC6A1 WT and KO MGEOs revealed downregulation of key MGE specification and cortical interneuron maturation genes, including LHX6, LHX8, SOX6, SHH, ARX, and DLX6, alongside upregulation of dorsal neuronal progenitor markers PAX6, ZIC2 and ZIC5. These results point to dysregulated MGE-derived interneuron development and accelerated or disrupted maturation with an initial bias toward early-born SST interneuron fates as well as a potential reduction of MGE identity with a ventral-to-dorsal fate shift.
Conclusions:
A delicate balance of ambient GABA levels during development is essential for proper GABAergic interneuron development. Our findings demonstrate that loss of SLC6A1 and subsequent GAT-1 deficiency disrupts key transcriptional programs required for MGE GABAergic cell specification and interneuron maturation, resulting in early SST overexpression, premature neuronal maturation, and reduced expression of core MGE markers. These alterations likely contribute to impaired interneuron development, potentially altering subsequent network formation. Further investigation into these phenotypes could provide mechanistic insights and treatment targets for SLC6A1-related neurodevelopmental disorders.Funding:
This work was supported by NIH (NINDS) U54NS117170 (JMP), the NSF Graduate Research Fellowship Program (MCV) and the NIH (NINDS) DSPAN F99/K00 predoc to postdoc transition award (MCV).