Abstracts

Rationale: Kcna1-null mice, lacking the delayed rectifier voltage-gated potassium channel α subunit Kv1.1, model temporal lobe epilepsy (TLE) and sudden unexpected death in epilepsy (SUDEP). We have previously shown that ketone bodies such as beta-hydroxybutyrate (BHB, produced during treatment with the high-fat ketogenic diet, or KD) exert anti-seizure effects through modulation of the mitochondrial permeability transition (mPT) pore, a multimeric protein complex on the mitochondrial membrane that when activated leads to release of cytochrome C and subsequent apoptosis/necrosis (PMID: 25899847). Specifically, it was demonstrated that BHB indirectly inhibits cyclophilin D (CypD, a regulatory subunit of the mPT complex) which prevents mPT opening. To show that blockade of mPT renders an anti-seizure effect; we created a double knockout of CypD and Kv1.1 and assessed seizure frequency and burden, as well as survivability. We hypothesized that the CypD/Kv1.1 double knockout mouse would experience fewer daily seizures, a lower seizure burden, and live longer than spontaneously epileptic Kcna1-null controls. Methods: We mated CypD-deficient (Ppif-null) mice on a B6;129 background with C3H wild-type (WT) mice, and then crossed the heterozygous progeny with C3H WT mice until the resultant CypD heterozygotes were ~97% pure with respect to the C3H background. The CypD heterozygotes were then mated to create CypD-deficient mice on a C3H background and the resultant mice lacking CypD were crossed with Kcna1-null heterozygotes yielding double heterozygotes. The double heterozygotes were crossed with CypD-null mice yielding both CypD-null and Kcna1 heterozygous mice which were mated to produce CypD/Kv1.1 double knockout mice. To assess seizure frequency and burden we utilized video-EEG monitoring. Animals were implanted with screw EEG electrodes and monitored for 10 days. A seizure detection algorithm was developed, using MATLAB, for rapid quantification. The algorithm calculates line-length, defined as the sum of distances between successive samples of the EEG. The line-length was z-scored, and two thresholds were set: one at 5 standard deviations above the mean line-length, to tag threshold crossings as seizures, and another, at 1 standard deviation above the mean, to indicate start and end times of a seizure. Seizures were verified manually by inspecting the video-EEG data, and assigned a modified Racine score. Seizure burden was calculated as the quotient of the summed Racine scores and the duration of the observation period. Results: CypD/Kv1.1 double knockout mice experienced fewer daily seizures (M=3.19, 95% CI [1.10, 5.27]; N=11) than Kcna1-null mice (M=7.10, 95% CI [3.73 10.47]; N=8); t(17) = 2.39, p=.028, including fewer convulsive (Racine>2) seizures (M=0.98, 95% CI [0.73, 1.23]; N=11), than Kcna1-null mice (M=2.93, 95% CI [1.61 4.25]; N=8); t(7.56) = 3.98, p=.001. In addition, CypD/Kv1.1 double knockout mice experienced lower seizure burden (Mdn= 4.24; N=11) than Kcna1-null mice (Mdn= 13.45; N=8), U=18.0, p=.032. Furthermore, the double knockout mice lived longer (Mdn=62.0 days; N=19) than Kcna1-null mice (Mdn=48.0 days; N=29); χ2(1) =17.34, p < .0001. Conclusions: Collectively, our data indicate that deletion of CypD can afford anti-seizure effects in epileptic Kcna1-null mice, and highlights the fact that targeted gene deletion can partly reverse the epileptic phenotype arising from absence of a critical potassium channel subunit. Moreover, our results are consistent with the notion that CypD is a novel target for epilepsy therapeutics. Funding: CIHR 10015062Alberta Children’s Hospital Research Institute Alberta Children’s Hospital Foundation NIH NS070261 Barrow Neurological Foundation.