Cellular and Gene Expression Changes in SCN8A Using Hippocampal Organoids
Abstract number :
1.032
Submission category :
1. Basic Mechanisms / 1B. Epileptogenesis of genetic epilepsies
Year :
2024
Submission ID :
600
Source :
www.aesnet.org
Presentation date :
12/7/2024 12:00:00 AM
Published date :
Authors :
Presenting Author: Anjana Shriram, BS (expected 6/2026) – University of California, Los Angeles
Colin McCrimmon, MD, PhD – University of California, Los Angeles
Marie Pahos, BS – University of California, Los Angeles
Daniel Toker, PhD – University of California, Los Angeles
Ranmal Samarasinghe, MD, PhD – University of California, Los Angeles
Rationale: Developmental and Epileptic Encephalopathy 13 (DEE13) is a severe neurodevelopmental disorder (NDD) associated with intractable, early-onset epilepsy and profound cognitive delays. This NDD typically results from a gain-of-function (GOF) mutation in the SCN8A gene encoding the Nav1.6 sodium channel. While prior studies have focused on the Nav1.6 properties and neuronal hyperexcitability in primarily cortical DEE13 models, here we studied the direct downstream effects of this GOF mutation on hippocampal cytoarchitecture and gene expression using an organoid platform that recapitulates features of developing human brains. Given the role of the hippocampus in SCN8A-related seizures1, learning, and memory, our findings are highly relevant for a better understanding of DEE13 pathology.
Methods: Induced pluripotent stem cells from one DEE13 patient with a pathogenic p.R1872 >L SCN8A variant (mut line), along with the CRISPR-corrected control (iCtrl line), were used to create organoids of hippocampal (Hc) and ganglionic eminence (GE) fates. These organoids were fused on day 56 to create Hc+GE “assembloids,” with rich excitatory neuron (ExN) and inhibitory neuron (InN) populations that promote complex network dynamics. At day 120, we performed immunohistochemistry (IHC) to analyze changes in ExN and InN populations (including SST- and RELN-expressing InN subtypes, that latter putative oriens-lacunosum moleculare (OLM) interneurons that play an integral role in phase-amplitude coupling (PAC) and hippocampal information flow). Single-nuclei RNA sequencing (snRNAseq) of several differentiations of mut and iCtrl assembloids was used to corroborate the IHC findings, and for differential gene expression and gene-ontology (GO) analyses.
Results: IHC revealed a significant increase in ExNs and a decrease in InNs, including a loss of SST- and RELN-expressing subtypes, in the mut assembloids. These exact changes have been shown to reduce PAC in a previously developed in-silico hippocampal model. The IHC findings were confirmed by snRNAseq, and GO analysis suggested that genes upregulated in mut assembloids were involved in excitatory/glutamatergic pathways, while downregulated genes were involved in basic cell functions.
Conclusions: This is the first study of cytoarchitectural and gene expression changes associated with a pathogenic SCN8A variant using a hippocampal organoid model. We demonstrated an increased ratio of excitatory to inhibitory neurons in mut DEE13 assembloids and a facilitation of excitatory pathways while sacrificing basic cellular functions that may produce an environment toxic to certain developing cells. This may explain the absence of interneuron subtypes such as OLM in mut assembloids, which in turn may play a role in the seizures and cognitive deficits associated with DEE13.
Reference: 1. Makinson CD, Tanaka BS, Lamar T, Goldin AL, Escayg A. Role of the hippocampus in Nav1.6 (Scn8a) mediated seizure resistance. Neurobiol Dis. 2014;68:16-25.
Funding: This work was supported by NINDS K08NS119747, Simons-BTI 717153, and CURE 20204012
Basic Mechanisms