Cholecystokinin 2 Receptor Antagonists for the Treatment of Temporal Lobe Epilepsy in Animal Models

Abstract

Epilepsy is a chronic disease of the central nervous system that affects approximately 39 million people worldwide, and one out of four epilepsy patients are resistant to drug treatment. New insights into the mechanism of epilepsy may facilitate the discovery of new anti-epileptic drugs to bring hope to refractory patients.

This dissertation adopted the kainic acid (KA)-induced temporal lobe epilepsy (TLE) model to investigate the neural circuits and molecular mechanisms of epilepsy. Injecting KA into the hippocampus causes a mouse to develop spontaneous seizures for the rest of its life. These seizures are typical focal-onset seizures. Epileptic activities originate from the temporal lobe, propagate to the bilateral neocortex and induce convulsive seizures. The propagation process was confirmed by our histological results that the early-gene c-fos was first displayed in the hippocampus and then spread to the neocortex. Next, a chemogenetic method was used to silence the entorhinal cortex (Ent) after KA infusion. We found that the c-fos intensity in the neocortex was reduced after suppressing the Ent, indicating the essential role of the Ent in the propagation of epileptic waves.

To further understand the process of seizure propagation, we measured the electrophysiological properties of the Ent in the acute phase of epilepsy induction. The power of the local field potential was remarkably increased after KA infusion, and the percentage of the high-frequency β- and γ-band was also raised. In addition, we found burst firings of single units after KA injection. High-frequency firing enables neuropeptide release. Our previous works also revealed that high-frequency stimulation of the Ent induces long-term potentiation in the neocortex, which is cholecystokinin (CCK) dependent.

Therefore, we queried whether the neuropeptide CCK and CCK⁺ neurons in the Ent are involved in epilepsy. To dig into this question, we adopted the transgenic methods to express GCaMP6s in principal CCK⁺ neurons in the Ent and recorded fluorescence dynamics with fiber photometry. We found that the spontaneous seizures dramatically activated the Ent CCK⁺ neurons, and epileptic mice sustained higher calcium dynamics during interictal phases. In addition, the qPCR experiments revealed that the CCK mRNA expression in the Ent of epileptic mice was higher than that of the control mice.

There are two endogenous CCK receptors in the brain: CCK1R and CCK2R; the latter shows a wide distribution in the neocortex. We hypothesized that CCK2R antagonists could suppress the strengthening of excitatory circuits and reduce the frequency of seizures. The hypothesis was verified by our pharmacological study on epileptic mice that CCK2R antagonist YF476 reduced the number of spontaneous convulsions. However, the brain of the mouse, which lacks sulci, fissures, and white matter, is quite distinct from the human brain. To further evaluate our drug candidates, we endeavored to develop a TLE model in miniature pigs.

By injecting KA into the right hippocampus, we established a TLE model in Bama miniature pigs, which successfully developed both convulsive and non-convulsive seizures after surgery. Afterward, we tested the anti-epileptic effects of YF476 and compared them with those of two anti-epileptic drugs, phenytoin and carbamazepine. Our results revealed that YF476 reduced spontaneous seizures in the mini-pig model of TLE: severe and mild seizures were reduced by 87% and 73% on average, respectively. The therapeutic effects were comparable to those of currently available anti-epileptic drugs.

To conclude, the present study provides substantial evidence to support the role of CCK and the Ent CCK⁺ neurons in epilepsy. First, we revealed that the Ent is essential for propagating epileptic activities from the hippocampus to the neocortex. It concerts with our second observation that seizures result in high-frequency activities of the Ent neurons, which is the prerequisite for CCK release. After that, we noticed that the principal CCK⁺ neurons in the Ent exhibit enhanced calcium activities after epilepsy induction. In addition, we examined the level of CCK mRNA expression and found that it was higher in the Ent of KA mice than that of control mice. After an effort to string these results together, we propose a possible mechanism underlying epilepsy that the upregulated CCK expression strengthens the excitatory connections to facilitate seizure propagation.

Importantly, this study initiated and successfully developed a TLE pig model. The therapeutic effects of the CCK2R antagonist YF476 in mice were also confirmed in mini-pigs, which sheds light on a potential drug candidate for epilepsy.

Date of Award	2 Jul 2021
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Jufang HE (Supervisor) & Micky TORTORELLA (External Co-Supervisor)