Ketone bodies mediate antiseizure effects through mitochondrial permeability transition

Abstract

Objective: Ketone bodies (KB) are products of fatty acid oxidation and serve as essential fuels during fasting or treatment with the high-fat antiseizure ketogenic diet (KD). Despite growing evidence that KB exert broad neuroprotective effects, their role in seizure control has not been firmly demonstrated. The major goal of this study was to demonstrate the direct antiseizure effects of KB and to identify an underlying target mechanism.

Methods: We studied the effects of both the KD and KB in spontaneously epileptic Kcna1-null mice using a combination of behavioral, planar multielectrode, and standard cellular electrophysiological techniques. Thresholds for mitochondrial permeability transition (mPT) were determined in acutely isolated brain mitochondria.

Results: KB alone were sufficient to: (1) exert antiseizure effects in Kcna1-null mice, (2) restore intrinsic impairment of hippocampal long-term potentiation and spatial learning-memory defects in Kcna1-null mutants, and (3) raise the threshold for calcium-induced mPT in acutely prepared mitochondria from hippocampi of Kcna1-null animals. Targeted deletion of the cyclophilin D subunit of the mPT complex abrogated the effects of KB on mPT, and in vivo pharmacological inhibition and activation of mPT were found to mirror and reverse, respectively, the antiseizure effects of the KD in Kcna1-null mice.

Interpretation: The present data reveal the first direct link between mPT and seizure control, and provide a potential mechanistic explanation for the KD. Given that mPT is increasingly being implicated in diverse neurological disorders, our results suggest that metabolism-based treatments and/or metabolic substrates might represent a worthy paradigm for therapeutic development.

Figure 1

A ketogenic diet (KD) and ketone bodies (KB) provide anti-seizure effects in epileptic Kcna1-null (KO) mice. (A) Interictal (upper three traces) and ictal EEG (bottom trace) recordings from wild-type (WT) and KO mice treated with either standard rodent diet (SD) or a KD. The bottom trace (KO/SD) depicts an electrographic seizure coincident with a behavioral generalized tonic-clonic seizure (N=12 in each group). (B) Mean daily tonic-clonic seizure frequency in KO mice over 3-day recording period between SD-treated vs. KD-fed animals, *, p<0.05. (C) Mean daily seizure frequencies over a 3-day recording period in KO mice after a 10–14 day treatment with either BHB or saline. Chronic osmotic mini-pump administration of BHB resulted in anti-seizure effects. One-way ANOVA followed by Tukey test; **, p<0.01. (D-F) Long-term exposure to KB decreases seizure-like events (SLEs) in organotypic slice cultures of KO hippocampi. (D) Light microscopic view of a hippocampal slice culture (14 days in vitro [DIV]) on a 64-microelectrode array. (E) Representative SLEs from a slice cultured for two weeks with either normal glucose or KB (5 mM BHB and 1 mM ACA). High-frequency oscillations (HFOs) revealed by band-pass filtering were present during SLEs in both culture conditions (lower traces). (F) KB significantly reduced SLE duration (~67%) and intensity (~80%) (n=4 cultures harvested from three P3–5 mice per group, unpaired t-test: *, p<0.05; **, p<0.001). For panels (B, C and F), data points reflect the mean ± SEM.

Figure 2

Metabolic parameters determined during the course of treatment. (A, B, C) Plasma β-hydroxybutyrate (BHB), blood glucose levels and weight changes in three cohorts of wild-type (WT) or Kcna1-null (KO) mice treated with either a standard diet (SD) or a ketogenic diet (KD). (D, E, F) Similar measurements made in separate groups of mice administered BHB through osmotic mini-pumps (N=12 mice in each group). Each symbol indicates the mean ± S.E.M; *p<0.05, **p<0.01. One-way ANOVA followed by Tukey test.

Figure 3

The cyclophilin D (CypD) subunit of the mitochondrial permeability transition (mPT) complex is required for functional neuroprotection by ketone bodies (KB). (A and B) Ketogenic diet (KD) treatment increases mPT threshold in Kcna1-null (KO) mice. Isolated brain mitochondria from wild-type (WT) C3H mice were able to accumulate more Ca²⁺ than mitochondria prepared from KO mice. (A) Representative spectrofluorometric traces indicating the capacity of mitochondria to sequester Ca²⁺ administered through bolus injections. Two weeks of KD treatment increased the capacity for Ca²⁺ uptake and fully restored mPT threshold in KO mitochondria. (B) Summary bar graphs of the different treatment groups. Each sample comprised pooled mitochondria from 3 mice. * p <0.05, n=4 per group. (C) β-hydroxybutyate (BHB) increases mPT threshold in acutely isolated cortical mitochondria from Kcna1-null (KO) mice. Graph indicates the different treatment groups normalized to KO mitochondria. For comparison, the mPT threshold in mitochondria from ketogenic diet (KD)-treated KO mice is shown (black bar). Each bar indicates the mean ± S.E.M, and each mitochondrial sample tested came from a single mouse. ** p<0.01, *** p<0.001, one-way ANOVA with post-hoc Bonferonni correction, n=4–5 samples per group. (D and E) Traces depicting thresholds for Ca²⁺-induced mPT in acutely isolated hippocampal mitochondria from B6 wild-type mice (D) and age-matched CypD-null mice (E). For these experiments, calcium was administered through micro-infusion. In CypD-null mice, neither BHB nor ACA – each 1 mM – were effective in raising the mPT threshold. Cyclosporin A (1 μM) was also not effective in altering Ca²⁺-induced mPT. (F) Summary bar graphs showing the mean ± S.E.M. of mPT thresholds for the various treatment groups. Note: the thresholds for calcium-induced mPT differed not only amongst the treatment groups, but also varied as a function of different background strain, and whether mice were wild-type or null mutants. (G) Ca²⁺ -induced swelling of control mitochondria with or without pre-incubation of 1 mM ACA and 1 mM BHB in control C57Bl6 mice. These experiments were performed under de-energized conditions and with a Ca⁺² ionophore in order to equilibrate this cation across the mitochondrial inner membrane. Ca⁺² (200 μM)-induced swelling increased in control, as well as ketone-treated samples. ACA and BHB treatment did not inhibit Ca⁺²-induced mitochondrial swelling as shown. Data represent n=5 animals in each group and samples were measured in two replicates per animal. (H) Atractyloside reverses the anti-seizure effects of the KD. Mean daily seizure frequencies over a 3-day recording period in Kcna1-null mice after various two-week treatments – those administered: (1) a standard diet (SD) only; (2) a KD plus atractyloside (ATRAC) 30 mg/kg IP daily; (3) a SD plus ATRAC; (4) a KD only; and (5) the selective cyclophilin D (CypD) inhibitor NIM811, 10 mg/kg IP daily. NIM811 resulted in an anti-seizure effect similar to what was observed with either the full KD or KB (see Figure 1). One-way ANOVA followed by Tukey test; **, p <0.01. N=8–9 per group. For panels (B, F, G and H), data points reflect the mean ± S.E.M.

Figure 4

The KD and KB induce nootropic (cognitive-enhancing) effects in epileptic brain. (A) Average time latencies for mice to find the escape chamber in the Barnes maze are summarized for three groups of mice: (1) wild-type (WT) mice fed a standard diet (SD); (2) Kcna1-null mice fed a SD; (3) Kcna1-null mice fed a KD. KD-treated Kcna1-null mice exhibited improved performance in the Barnes maze (as indicated by the shorter latencies) compared to Kcna1-null mice fed only a SD. Interestingly, Kcna1-null mice fed the KD performed as well or better than WT mice fed the SD. N=18–24 per group *, p<0.05. (B) Kcna1-null mice fed a KD exhibit restoration of LTP induction after a two-week treatment period (red circles; N=16 slices from 6 mice). Standard diet (SD)-fed Kcna1-null mice show impairment of LTP induction (open circles; N=13 slices from 6 mice) compared to WT mice fed regular rodent chow (black circles). (C) Chronic in vivo BHB administration for one week via osmotic mini-pumps results in restoration of intrinsically impaired hippocampal LTP in Kcna1-null mice (N=14 slices from 8 mice). (D) ATRAC treatment negates the protective effects of the KD on intrinsic impairment of CA1 hippocampal long-term potentiation (LTP) induced by high-frequency stimulation (HFS; 100 Hz, 1 sec) of Schaffer collaterals. ATRAC alone does not influence the EPSP amplitude after HFS in Kcna1-null mice. N=12–14 slices from 6–8 mice in each group. (E) NIM811 – similar to either the KD or beta-hydroxybutyrate (BHB) alone restores intrinsic impairment of hippocampal LTP (n=10–16 slices from 4–7 mice in each group). (F) Summary data of EPSP amplitudes 1 hour after LTP induction for various treatment groups. For all panels, data points and bars reflect the mean ± SEM. **, p<0.01.

Figure 5

Comparison between the ketogenic diet (KD) and phenobarbital (PB) on spontaneous recurrent seizures, hippocampal long-term potentiation (LTP) and mPT thresholds. (A) Seizure frequency in Kcna1-null (KO) mice over a 3-day recording period after anti-seizure treatments. Both the KD and PB (30 mg/kg/d IP daily for 10 days) resulted in a significant decrease in seizure frequency compared to standard diet (SD) treatment, and were similarly effective (N=9 in each group). (B) PB worsened LTP maintenance in WT mice, and did not confer protective effects on the EPSP amplitude in Kcna1-null animals. PB (30 mg/kg daily) was administered IP over 10 days. Each set of traces was derived using 16 hippocampal slices from 9 mice. (C) Representative traces demonstrating that PB (100 μM) did not significantly alter the threshold for Ca⁺²-induced mPT in acutely isolated mitochondria from C57/Bl6 mice, compared to the control compound 1 μM CsA. (D) Summary bar graph reflecting the means ± S.E.M. of mPT thresholds in the different treatment groups. N=10–13 separate runs in each group from mitochondria prepared from 9 mice. *, p<0.05, ***, p<0.001; N.S., not significant. One-way ANOVA followed by Tukey test.