Ketogenic diet-induced extension of longevity in epileptic Kcna1-null mice is influenced by gender and age at treatment onset

Abstract

INTRODUCTION

Sudden unexpected death in epilepsy (SUDEP) is a clinically significant problem – affecting as many as 1 per 1000 patients annually (Maguire et al., 2016; Surges et al., 2009). The clinical hallmark feature of SUDEP appears to be cardiorespiratory compromise associated with a terminal generalized tonic-clonic (GTC) seizure (Surges et al., 2009). To date, several risk factors for SUDEP have been identified, including GTC frequency, gender, early age at seizure onset and polytherapy with anti-seizure drugs (ASDs) (Maguire et al., 2016; Nilsson et al., 1999). Given the profound impact of SUDEP in patients with medically intractable epilepsy, the development of therapeutic strategies to prevent this catastrophic outcome is of paramount importance.

Among the few clinically relevant animal models of SUDEP is the Kcna1-null mutant (KO) mouse lacking the delayed rectifier potassium channel α subunit Kv1.1 (Glasscock et al., 2010; Simeone et al., 2016). These KO mice manifest severe spontaneous recurrent seizures (SRS) beginning early in postnatal development and experience premature sudden death. Recently, the high-fat ketogenic diet (KD), a clinically proven therapy for patients with treatment-resistant epilepsy, was shown to extend lifespan in KO mice (Simeone et al., 2016). To delineate specific experimental variables impacting this protective effect, we asked whether gender and/or age at initiation of dietary treatment might affect longevity.

METHODS

RESULTS

KD administration affords seizure suppression and lifespan extension in KO mice

A growing body of evidence has supported the anti-seizure effects of the KD in epileptic brain (Kim et al., 2015). Given that deletion of the Kv1.1 potassium channel protein results in spontaneous recurrent seizures (SRS) and death early in life (Kim et al., 2015; Simeone et al., 2016), we asked whether the KD might also affect the early mortality. In our hands, SD-fed KO mice experienced sudden unexpected death at PD 46.9 ± 0.8 (n=112) (Fig. 1A). As mice grew older, KO mice exhibited an increase in SRS, with a daily seizure frequency of 12 ± 2.8 at PD40-45 (Fig. 1C). When KO mice were treated with the KD starting at PD30, the mean daily seizure frequency decreased to 4.1 ± 0.9 between PD40-45, and the average age at the time of sudden death was prolonged to PD69.8 ± 1.7 (n=36) (Fig. 1B &C). Nonetheless, despite an overall lower seizure frequency in KD-fed KO mice during aging, the KD failed to block a terminal GTC-driven sudden death.

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Figure 1

Ketogenic diet (KD)-induced longevity extension in Kcna1-null (KO) mice. (A) There are no gender differences in SUDEP risk in SD-fed KO mice. (B) Following KD treatment starting at PD30, KD-fed KO mice survived longer than SD-fed KO mice, and this effect was more pronounced in KD-fed female KO mice compared to male counterparts. (C) Summary of daily seizure frequencies in KO mice with or without the KD treatment between PD40-45. (D) Summary bar graph of the different experimental groups showing sex-dependent lifespan effects. (E) Summary data indicating effects on lifespan of KO mice as a function of age at initiation of the KD (PD25 vs. PD35) and gender. (F) KD-fed KO mice exhibited significant increases in blood BHB levels compared to SD-fed mice (N=7 per group; ** p <0.01). The pound sign reflects significant differences between KD-fed female mice and KD-fed male mice (p < 0.05). Each symbol indicates the mean ± standard error of the mean. One-way analysis of variance followed by Tukey test or t-test: *, ^# p < 0.05, ** p <0.01, *** p <0.001 and NS, not significant.

DISCUSSION

The principal finding of this study is that KD treatment significantly prolongs the lifespan of epileptic KO mice, and with a concomitant reduction in seizure activity and consistent ketonemia. Despite lack of direct clinical evidence about the efficacy of the KD in SUDEP in humans, these results are consistent with a recent report demonstrating the beneficial effects of the KD in the same animal model of SUDEP (Simeone et al, 2016). The current study extends this earlier finding by demonstrating that female KO mice live longer in response to the KD, and exhibit higher levels of ketosis, relative to comparably aged male epileptic animals. Further, the lifespan extension induced by the KD is more pronounced when dietary treatment is started at an earlier age. Collectively, our data support the notion that metabolism-based treatments such as the KD might represent a readily available avenue to help prevent sudden death in epilepsy and highlight two factors that could potentially influence outcome.

Although earlier retrospective clinical data addressing the interaction between gender and sudden unexpected death risk indicated that male patients were more susceptible to SUDEP (Nilsson et al., 1999), there were no sex differences in our KO mice fed a SD. However, lifespan in KO mice was extended to a greater degree when the KD was administered earlier in life, and this effect was more pronounced in female animals. The underlying basis for this observation remains unclear, but it is possible that this is due to a better response in terms of seizure improvement to the KD. Consistent with this notion is a recent retrospective clinical study that reported improved seizure control in female, compared to male, patients while on the KD (Agarwal et al, 2017). This potential sex difference requires further investigation since previous clinical observations failed to reveal gender as a critical factor in seizure control with the KD (Schoeler et al., 2013).

One interesting observation in the present study is that differences in longevity of KD-fed KO mice based on gender correlated with BHB levels. It is intriguing to speculate that higher levels of BHB may afford better seizure control (Kim et al., 2015), although the relationship between ketone levels and the clinical efficacy of the KD remains unclear. Furthermore, there has been growing interest in the effects of BHB on longevity in general (Veech et al., 2017). Lifespan prolongation may involve epigenetic mechanisms, such as modulation of histone deacetylases, mammalian target of rapamycin (mTOR), and/or sirtuins (McDaniel et al., 2011; Shimazu et al., 2013; Moore et al., 2014; Zhao et al., 2017).

One-third of individuals with epilepsy continue to have uncontrolled seizures despite taking multiple ASDs. In this population of patients, both early age of seizure onset and polytherapy with AEDs have been identified as potential risk factors for SUDEP (Maguire et al., 2016; Nilsson et al., 1999). In addition, there are genetic risk factors for premature sudden death in epilepsy. For example, mutations in the human KCNA1 gene encoding the Kv1.1 channel confers increased risk (Klassen et al., 2014), and loss of one allele may pose an intermediate risk, as evidenced by heterozygous Kv1.1 mice having a higher predisposition for seizures (Rho et al., 1999). However, following KD treatment beginning at both PD25 and PD35, we found that Kcna1-null mice showed enhanced longevity, but this beneficial effect was more striking in group started at PD25. Since PD25-35 in mice may be equivalent to human age range between 6 months and less than 10 years (Dutta et al., 2016), this age-dependent outcome is of potential clinical importance. Further clinical studies addressing mortality rates and seizure control in patients with SUDEP with and without KD treatment are necessary to validate our experimental findings.

Although the pathophysiological mechanisms underlying SUDEP remain unclear, one possibility is that seizure activity could spread into the medullary respiratory and autonomic centers, precipitating cardiac bradyarrhythmias due to increased parasympathetic tone (Glasscock et al., 2010; Moore et al., 2014). It is unknown whether KD administration influences SUDEP-related cardiac dysrhythmias, but long-term treatment with this diet in children with medically intractable epilepsy does not appear to induce detrimental effects on ventricular function (Ozdemir et al., 2016). Further, there is growing evidence that the KD imparts potential cardioprotective effects against ischemic insults as well as longevity extension, possibly through regulation of mitochondrial redox state and phosphorylation (Al-Zaid et al., 2007; Krebs et al., 2011).

In summary, our data further support the notion that the KD can prolong survival of epileptic Kcna1-null mice (that have previously been shown to expire from SUDEP), and that this effect is dependent on sex and age at initiation of therapy. The profound alterations in cellular metabolism and mitochondrial bioenergetics elicited by the KD are likely to be influenced by other factors as well, and future studies are necessary to clarify the specific mechanisms underlying the disease-modifying effects of the KD. Greater fundamental knowledge about KD-driven longevity extension will then critically inform novel therapeutic approaches to prevent SUDEP.

Acknowledgments

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