Authors :
Presenting Author: Yosuke Miyamoto, MD – Kyoto Prefectural University of Medicine
Eisuke Ichise, MD, PhD – Kyoto Prefectural University of Medicine
Amane Matsuura, MD – Kyoto Prefectural University of Medicine
Satoshi Yamashita, MD, PhD – Kyoto Prefectural University of Medicine
Takenori Tozawa, MD, PhD – Kyoto Prefectural University of Medicine
Tatsuji Hasegawa, MD, PhD – Kyoto Prefectural University of Medicine
Masafumi Morimoto, MD, PhD – Kyoto Prefectural University of Medicine
Mitsuru Ishikawa, PhD – Fujita Health University School of Medicine
Tomohiro Chiyonobu, MD, PhD – Kyoto Prefectural University of Medicine
Rationale:
AKT3, a key component of the PI3K-AKT-MTOR pathway, is highly expressed in the brain, and its gain-of-function variants cause megalencephaly and cortical malformations, often associated with intractable epilepsy. Capivasertib, a selective pan-AKT kinase inhibitor, was developed for patients with advanced solid malignancies. We postulated that it holds potential as a targeted therapy for epilepsy caused by AKT3 activating variants. In this study, we generated iPSC-derived glutamatergic neurons from a patient harboring a germline AKT3 activating variant and evaluated the efficacy of capivasertib from the perspective of epilepsy treatment.
Methods:
iPSC lines derived from a heterozygous AKT3 variant (p.Q78R) patient were established from monocytes. Isogenic controls were generated using the CRISPR/Cas9 system (Figure 1A). For glutamatergic neuron induction, NEUROG2–transfected cells were cultured under optimized conditions (Figure 1B). The phosphorylation state of S6, a downstream component of the PI3K-AKT-MTOR signaling, was examined 4 weeks after neuronal differentiation by western blotting. Immunostaining was performed at week 4 of neuronal differentiation, and cell soma size was evaluated using ImageJ. Electrophysiological activities were recorded weekly at 4–6 weeks after neuronal differentiation using a microelectrode array. Cells were exposed to capivasertib at 0.1, 0.3, or 1 μmol/L for 2 weeks from week 4 of neuronal differentiation.
Results:
Patient-derived neurons had larger somas and exhibited an increase in phospho-S6 signals compared to isogenic controls. Patient-derived neurons showed a significant increase in the number of bursts at 5 weeks after neuronal differentiation. At 6 weeks of differentiation, the number of total spikes and firing rate for each active electrode of the patient lines were also increased. Exposure to patient-derived neurons to capivasertib at concentrations of 0.3 or 1 μmol/L suppressed the number of total spikes and bursts.
Conclusions:
We demonstrated for the first time that spontaneous electrical activity is increased in patient iPSC-derived glutamatergic neurons of AKT3-related megalencephaly syndrome, indicating that this model provides a valuable platform for evaluating therapeutic strategies for epilepsy associated with the disorder. Capivasertib inhibits abnormal spontaneous firing activity of patient-derived neurons, suggesting its potential application in the treatment of epilepsy. This study suggests that such a drug repurposing approach could be an effective strategy for addressing rare and hard-to-treat forms of epilepsy.
Funding:
This work was supported by JSPS KAKENHI (23K07339, 25K11085), the Japan Epilepsy Research Foundation (25008), and the Tokumori Yasumoto Memorial Trust for Researches on Tuberous Sclerosis Complex and related Rare Neurological Disease.