Abstracts

Rationale: Most cases of absence epilepsy do not show a familial pattern of inheritance, suggesting that the heredity of absence epilepsy is polygenic. Many sporadic absence epilepsy patients carry heterozygous mutations in the CACNA1H gene that codes for the T-type calcium channel subunit alpha1H (α1H). Therefore, mutations in the CACNA1H gene are associated with absence epilepsy heredity and warrant further investigation. We recently demonstrated that loss of function Cacna1h gene mutation promotes absence seizure generation when deleted in a tamoxifen-induced adult Cacna1a gene knock-out model (CAG-CreER;Cacna1a^flox/flox;Cacna1h^-/-) (Miao et al, 2020). The Cacna1a gene encodes the P/Q-type calcium channel subunit α1A. Homozygous, but not heterozygous, loss of Cacna1a gene produces absence epilepsy in mice, which is rare in human patients. No genetic animal model has been established to mimic human absence epilepsy via well-defined polygenic inheritance. To this end, I hypothesized that combined heterozygous loss of Cacna1a and Cacna1h genes results in absence epilepsy.

Methods: CAG-CreER;Cacna1a^+/flox;Cacna1h^+/- and CAG-CreER;Cacna1a^+/flox;Cacna1h^-/- mice were created by crossing CAG-CreER;Cacna1a^flox/flox with Cacna1h^-/- mice. Genotype was validated by polymerase chain reaction (PCR). To induce Cre recombinase activity, adult mice ( > postnatal day 42) were injected once daily with tamoxifen [dissolved in corn oil, 75 mg/kg body weight, intraperitoneally (i.p.)] for five consecutive days. We recorded video-EEG before induction and on serial days post-first injection (DP1i) (DP1i7, DP1i14, DP1i21 and DP1i28). Ablation of Cacna1a is confirmed by immunohistochemistry (Anti-GFP, Abcam, Cat# ab6673, 1:500). Ethosuximide (i.p., 200 mg/kg body weight) was used to block absence seizures.

Results: Here we report that heterozygous loss of Cacna1a and Cacna1h results in absence epilepsy in mice (CAG-CreER;Cacna1a^+/flox;Cacna1h^+/-). Video-EEG recordings showed that they developed absence epilepsy characterized by spike-wave-discharge on EEG accompanied by behavioral arrest after tamoxifen induction (DP1i21), demonstrating an epileptogenic role of combined loss of Cacna1a and Cacna1h genes. Power spectral density analysis showed that the spike-wave-discharge in these mice at DP1i21 has similar dominant frequency (peaked around 5 Hz) to that observed in Cacna1a adult knockout mice (Miao et al, 2020). Systemic ethosuximide treatment blocked the absence seizure in them. Further removal of the other copy of the Cacna1h gene (CAG-CreER; Cacna1a^+/flox;Cacna1h^-/-) sustains the absence epilepsy phenotype, while we found that mice with heterozygous or homozygous loss of Cacna1h gene alone do not have absence seizure.

Conclusions: The Cacna1a and Cacna1h doubly heterozygous mice described here are the first genetic animal model to reveal the complex inheritance of absence epilepsy induced by distinct calcium channel mutations. This represents a transformative approach to model the intricate heredity of human absence epilepsy, which could provide a fundamental basis for discoveries leading to novel and effective therapeutic interventions in the future.

Funding: Please list any funding that was received in support of this abstract.: NS29709 (NINDS).