Rationale:
Sleep and circadian rhythm disturbances are hallmark comorbidities in epilepsy, particularly in severe genetic epilepsies like Dravet syndrome. However, the molecular underpinnings remain poorly understood, and no human in vitro model has yet been developed to study circadian rhythm disruptions in epilepsy. The aim is to establish the first human stem cell–derived model capable of capturing circadian rhythmicity and its disruption in Dravet Syndrome.
Methods: Human iPSC-derived neuron-astrocyte co-cultures were matured for 35 days. Three cell lines were used: Dravet syndrome (DS) patient-derived iPSCs, isogenic controls with corrected
SCN1A mutation, and healthy control iPSCs. Circadian rhythms were synchronised using a 2-hour serum shock, which models systemic entrainment signals from the suprachiasmatic nucleus (SCN) and is widely used to initiate circadian cycling in vitro. Circadian time zero (CT0) was defined as the start of serum exposure, with cultures subsequently sampled every 4 hours over 24 hours (CT4–CT24). Expression of 24 genes—including core circadian regulators (
BMAL1,
CLOCK,
NPAS2,
PER1–3,
CRY1–2) and associated transcriptional modulators—was quantified using high-throughput RT-qPCR. Network-level electrophysiological activity was assessed using multielectrode array (MEA) recordings, capturing circadian changes in spiking and bursting patterns. Gene expression and neuronal network activity were evaluated at baseline and following seizure induction using 4-aminopyridine (4-AP; 5 mM, 10 min) in DS, isogenic, and healthy control cultures.
Results: Neuron-astrocyte co-cultures showed robust circadian oscillations, with activator genes peaking at CT12 and repressors at CT4—rhythms absent in iPSCs and phase-delayed in neuron-only cultures. SCN1A epilepsy and 4-AP-induced activity both led to consistent disruption of circadian gene oscillations, including suppression of CLOCK, BMAL1, and PER1-3 peaks, and time-dependent upregulation of NPAS2. MEA recordings indicated parallel disruption in electrophysiological rhythms.
Conclusions: This is the first demonstration of a human iPSC-derived culture system that models molecular and electrophysiological circadian rhythms in epilepsy. Our findings reveal conserved disruptions in core clock gene dynamics across both genetic and acute seizure models, supporting the hypothesis that epilepsy perturbs fundamental circadian machinery. This platform opens new avenues to dissect circadian pathophysiology in epilepsy and guide future development of time-targeted (chronotherapeutic) interventions.
Funding:
Austrlaian Medical Research Future Fund (MRFF 2015957)