Project 1: High Throughput Functional Evaluation of Ion Channel Variants in Epilepsy
Project 2: Investigation of human neuron models of channelopathy-associated epilepsy
Project 3: Development and investigation of murine models of channelopathy-associated epilepsy
Core A: Variant Prioritization and Curation
Evangelos Kiskinis, Ph.D.
Owen McManus, Ph.D.
The cellular mechanisms responsible for channelopathy-associated epilepsy, including how intrinsic ion channel dysfunction cause paroxysmal defects in human neuron excitability, remain unclear. Recent advances in the generation of patient-specific induced pluripotent stem cells (iPSCs) offer new opportunities for elucidating pathogenic mechanisms of these disorders. Certain unique capabilities of iPSC technologies are well suited for the study of epilepsy, including: a) facilitating study of a disease in the context of each person's unique genetic background; b) enabling investigations of distinct human neuronal subtypes; and c) providing a platform for examining the effects of approved drugs or investigational compounds on neuron excitability and development. Project 2 exploits our expanding collection of validated, patient-specific iPSC lines from subjects with channelopathy-associated epilepsy involving SCN2A and KCNQ2 variants, along with corresponding clinical profiles including drug responses. Hence, we are uniquely positioned to use this powerful technology without new patient recruitment efforts.
Project 2 utilizes the collective expertise of the Center in iPSC technologies, genome editing, neuronal differentiation, pharmacology, traditional electrophysiology and an innovative, high-throughput optical platform (Optopatch) for assessing neuron excitability, to accelerate our understanding of the impact of ion channel variants on human neuron function. In Aim 1, we are examining cortical neurons derived from patient-specific iPSCs and respective isogenic control lines to elucidate the mechanisms responsible for abnormal excitability at the cellular and neuronal network levels. The specific variants represented in the patient-derived iPSCs will be adjudicated as high priority by Core A. We are also combining transcriptional profiling (single-cell RNA-Sequencing) with electrophysiological approaches (whole cell patch clamp recording and Optopatch) to interrogate iPSC-derived GABAergic interneurons and excitatory glutamatergic neurons, cultured separately or together. We employ voltage-clamp recording to measure and characterize M-current and Na-current for comparison with heterologous cell experiments performed on the same variants by Project 1. Results from experiments to interrogate neuron excitability will be correlated with findings from mouse models of identical variants investigated by Project 3 to determine the reliability and accuracy of iPSC technology to predict in vivo physiology and pharmacology.
In Aim 2, we are investigating the utility of using patient-specific iPSC-neuronal models of channelopathy-associated epilepsy for predicting optimal drug responses. These studies focus on iPSC lines from patients with severe neonatal onset epileptic encephalopathy associated with KCNQ2 and SCN2A variants. We are employing traditional electrophysiological approaches and Optopatch to assess intrinsic excitability and synaptic function, before and after treatment with NaV channel blockers and KV7 channel modulators that have clinical efficacy in the subjects from whom the cells were derived. Our goal is to rank the in vitro effectiveness of drugs in restoring normal neuron excitability for each genetic variant, and then to correlate the in vitro drug responses with the clinical responses to AEDs documented for these subjects.
Project 2 investigators interact with Project 1 to compare biophysical properties of variant ion channels in heterologous cells and iPSC-derived human neurons. Collaboration with investigators in Project 3 also enables side-by-side comparisons of neurophysiological and drug responses of human and mouse neurons carrying the same ion channel variant. These experiments will allow us to assess how an in vitro human neuron model system correlates with findings from heterologous expression systems and in vivo mouse models of disease that will be developed in parallel. Our efforts will elucidate the effects of ion channel variants on human neuronal excitability, provide a bridge between understanding the biophysical dysfunction of an ion channel with its cellular impact, and optimize a scalable model system for targeted pharmacological studies with translational value for improving drug selection for patients with channelopathy-associated epilepsy.
Highlights of Project 2:
• Availability of unique collection of iPSC lines
• Integration of electrophysiological and transcriptional readouts
• Industrial scale optical electrophysiology platform (Optopatch)
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Forrest MP, et al. 2023. Rescue of neuropsychiatric phenotypes in a mouse model of 16p11.2 duplication syndrome by genetic correction of an epilepsy network hub. Nat Commun (Feb 2023)
Ruggiero SM, et al. 2023. The current landscape of epilepsy genetics: where are we, and where are we going? Curr Opin Neurol (Apr 2023)
Clatot J, et al. 2023. A KCNC1-related neurological disorder due to gain of Kv3.1 function. Ann Clin Transl Neurol (Jan 2023)
Parthasarathy S, et al. 2022. A recurrent de novo splice site variant involving DNM1 exon 10a causes developmental and epileptic encephalopathy through a dominant-negative mechanism. Am J Hum Genet (Dec 2022)