Authors :
Presenting Author: Daniel Toker, PhD – University of California, Los Angeles
Colin McCrimmon, MD/PhD – University of California, Los Angeles; Kevin Lozano, B.S. – Neurology – University of California, Los Angeles; Marie Pahos, B.S. – Neurology – University of California, Los Angeles; László Molnár, PhD – Electrical Engineering – Sapientia Hungarian University of Transylvania; Istvan Mody, PhD – Neurobiology – University of California, Los Angeles; Jack Parent, MD – University of Michigan, Ann Arbor; Bennett Novitch, PhD – Neurobiology – University of California, Los Angeles; Ranmal Samarasinghe, MD/PhD – Neurology – University of California, Los Angeles
Rationale:
Pathogenic mutations in the SCN8A (Nav1.6) sodium channel can lead to developmental epileptic encephalopathy-13 (DEE-13), a condition linked with seizures, developmental delays, and intellectual disability. Given the high prenatal expression of Nav1.6, these mutations likely affect the early development of neural circuitry. However, studying human prenatal neural development remains challenging. An emerging solution to this challenge is the development of human brain organoids, which are 3D in vitro structures derived from human induced pluripotent stem cells (hiPSCs). These organoids recapitulate many functional features of the human brain, making them ideal platforms for studying early brain development. We have developed an "assembloid" organoid model in which excitatory hippocampal-like (Hc) and inhibitory ganglionic eminence-like (GE) neuron populations integrate. This fused organoid displays a mix of excitatory and inhibitory neurons, producing neural oscillations similar to those observed in the living hippocampus.
Methods:
Brain organoids were created from p.SCN8A mutant or matched control hiPSCs following standard laboratory protocols. These organoids were fused into assembloids on day 56. At around day 110, they were treated with AAV-1-GcAMP and then examined using two-photon microscopy by approximately day 124. Extracellular local field potentials (LFPs) were recorded when assembloids were age 120-130. Standard lab methods were used for Immunohistochemical (IHC) analysis to determine cell-type expression and the structure of the organoid.
Results:
Hippocampal and GE assembloids harboring the pathogenic SCN8A variant did not demonstrate overt hyperexcitability, but IHC analysis revealed a marked reduction in their SST+ interneuron population along with an increase in their excitatory neuron population compared to CRISPR-corrected controls. Moreover, LFPs in DEE-13 organoids revealed irregular phase-amplitude coupling patterns between theta (3-10 Hz) and gamma (30-120 Hz) frequencies compared to CRISPR-corrected assembloids. By leveraging recently developed nonlinear dynamics analysis tools applied to our LFP and two-photon calcium imaging recordings, as well as detailed biophysiological hippocampal circuit simulations, we determined that these irregularities stemmed from destabilized theta oscillations in SCN8A mutation-affected hippocampal assembloids.
Conclusions:
Given the critical role of stable theta oscillations and theta-gamma coupling in hippocampal encoding, our findings suggest a potential mechanism for the learning and cognitive deficits experienced by DEE13 patients.
Funding:
This work was supported by NINDS K08NS119747, Simons-BTI 717153, and Cure 20204012 (PI, Samarasinghe)