The Gut Microbiota Mediates the Anti-Seizure Effects of the Ketogenic Diet
Christine A. Olson,1 Helen E. Vuong,1 Jessica M. Yano,1 Qingxing Y. Liang,1 David J. Nusbaum,1 and Elaine Y. Hsiao1,2,*1Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, CA 90095, USA
The ketogenic diet (KD) is used to treat refractory ep- ilepsy, but the mechanisms underlying its neuropro- tective effects remain unclear. Here, we show that the gut microbiota is altered by the KD and required for protection against acute electrically induced sei- zures and spontaneous tonic-clonic seizures in two mouse models. Mice treated with antibiotics or reared germ free are resistant to KD-mediated seizure pro- tection. Enrichment of, and gnotobiotic co-coloniza- tion with, KD-associated Akkermansia and Parabac- teroides restores seizure protection. Moreover, transplantation of the KD gut microbiota and treat- ment with Akkermansia and Parabacteroides each confer seizure protection to mice fed a control diet. Alterations in colonic lumenal, serum, and hippo- campal metabolomic profiles correlate with seizure protection, including reductions in systemic gamma- glutamylated amino acids and elevated hippo- campal GABA/glutamate levels. Bacterial cross- feeding decreases gamma-glutamyltranspeptidase activity, and inhibiting gamma-glutamylation pro- motes seizure protection in vivo. Overall, this study re- veals that the gut microbiota modulates host meta- bolism and seizure susceptibility in mice.
The low-carbohydrate, high-fat ketogenic diet (KD) is an effec- tive treatment for refractory epilepsy, a condition affecting more than one-third of epileptic individuals and defined by a failure to respond to existing anticonvulsant medications (Kwan and Bro- die, 2000). However, despite its value for treating epilepsy and its increasing application to other disorders, including autism spectrum disorder, Alzheimer’s disease, metabolic syndrome, and cancer (Stafstrom and Rho, 2012), use of the KD remains low due to difficulties with implementation, dietary compliance, and adverse side effects (Freeman and Kossoff, 2010). Molec- ular targets are needed to develop viable clinical interventions for intractable epilepsy and other disorders for which the KD is beneficial. Many studies have proposed roles for ketone bodies, gamma-aminobutyric acid (GABA) modulation, and
mitochondrial anaplerosis in mediating the neurological effects of the KD (Rogawski et al., 2016), but exactly how the KD con- fers beneficial effects on brain activity and behavior remains unclear.
The gut microbiota is a key intermediary between diet and host physiology; the species composition and function of the gut mi- crobiota is critically shaped by diet, and nutrients made available to the host depend on microbial metabolism (Sonnenburg and Ba ̈ckhed, 2016). Diet-induced changes in the gut microbiota are reproducible and persistent (David et al., 2014), and as such, have lasting impacts on the host. Several diet-induced host pathologies are mediated by changes in the gut microbiota in mouse models, including symptoms of atherosclerosis in response to the carnitine-rich diet, undernutrition in response to the Malawian diet, and abnormal social behavior in response to maternal high-fat diet (Buffington et al., 2016; Koeth et al., 2013; Smith et al., 2013).
The gut microbiota modulates several metabolic and neuro- logical pathways in the host that could be relevant to KD- mediated seizure protection. The KD alters the composition of the gut microbiota in mice (Klein et al., 2016; Newell et al., 2016), and ketosis is associated with altered gut micro- biota in humans (David et al., 2014; Duncan et al., 2008). Inter- estingly, fasted mice that lack microbiota exhibit impaired he- patic ketogenesis and altered myocardial ketone metabolism compared to fasted mice that are conventionally colonized (Crawford et al., 2009). The microbiota is also increasingly associated with changes in factors relevant to neurotransmis- sion, including neurotransmitter signaling, synaptic protein expression, long-term potentiation, and myelination, as well as a variety of complex host behaviors, including stress- induced, social, and cognitive behaviors (Vuong et al., 2017). Notably, several clinical studies report that antibiotic treatment increases risk of status epilepticus or symptomatic seizures in epileptic individuals (Sutter et al., 2015), suggest- ing a possible role for the microbiota in mitigating seizure likelihood.
Based on emerging studies linking the gut microbiota to host responses to diet, metabolism, neural activity, and behavior, we hypothesized that the gut microbiota impacts the anti-seizure effects of the KD. We show herein that the gut microbiota is necessary and sufficient for seizure protection in two mouse models of intractable epilepsy and further identify cooperative interactions between two diet-associated bacteria that regulate levels of circulating dietary metabolites, brain neurotransmitters, and seizure incidence in mice.
The microbiota plays a key role in host digestion, metabolism, and behavior, but whether microbial responses to diet also
impact neuronal activity is poorly understood. Here, we demon- strate that the KD alters the gut microbiota across two seizure mouse models, and changes in the microbiota are necessary and sufficient for conferring seizure protection. Several clinical studies link antibiotic treatment to increased risk of status epi- lepticus or symptomatic seizures in epileptic individuals (Sutter et al., 2015). Prolonged treatment with metronidazole can pro- voke convulsions (Beloosesky et al., 2000), and ampicillin expo- sure is associated with elevated seizure risk (Hornik et al., 2016). Penicillin and other b-lactams are hypothesized to directly reduce GABAergic inhibition, but whether microbiota depletion contributes to increases in seizure frequency is not clear. Results from our study reveal that microbiota depletion via high-dose antibiotic treatment raises seizure susceptibility and incidence in response to the KD in both wild-type and Kcna1/mice. These effects of antibiotic treatment are abrogated by re-coloni- zation with gut bacteria, suggesting that links between antibiotic use and seizure incidence in humans could be mediated by the microbiota. Future investigation is warranted to determine whether human epilepsy is associated with microbial dysbiosis and whether antibiotic exposure in epileptic individuals impacts response to the KD.
We observe in both Taconic Swiss Webster and Jackson C3HeB/FeJ Kcna1/mice that the KD reduces gut bacterial alpha diversity, while elevating relative abundance ofA. muciniphila and Parabacteroides. Similar diet-induced in- creases in A. muciniphila are observed during fasting in humans (Dao et al., 2016; Remely et al., 2015). A. muciniphila and Para- bacteroides are also associated with increased ketosis (David et al., 2014) and metabolic improvement in humans (Everard et al., 2013). One study reports changes in the gut microbiota in response to the KD in BTBRT+tf/ and C57BL/6J mice, where levels of Akkermansia were correlated with levels of serum gluta- mate, lactate, taurine, and sarcosine (Klein et al., 2016). How- ever, different taxonomic shifts were observed, highlighting that the KD-induced microbiota likely depends on host genetics and baseline microbiota profiles. Indeed, different species, strains, and even cohorts of animals are known to exhibit micro- biota profiles that vary in taxonomic membership but are func- tionally redundant, raising the question of whether there are additional taxa that also perform similarly to A. muciniphila andParabacteroides in our study. Further research is needed to determine effects of the KD on microbiome profiles in individuals with refractory epilepsy and whether particular taxonomic changes correlate with seizure severity.
Amino acids are transported across the blood-brain barrier (BBB) and serve as nitrogen donors for glutamate and GABA biosynthesis (Yudkoff et al., 2001). GG-amino acids, in particular, are reported to exhibit increased transport properties, where the gamma-glutamyl moiety promotes translocation across lipid barriers (Castellano and Merlino, 2012). Our data suggest that KD- and microbiota-related restrictions in GG-amino acids are important for seizure protection, which aligns with previous studies linking GGT activity to altered seizure severity. In a study of 75 epileptic patients, high serum GGT activity was observed in 84.5% of the patients compared to controls (Ewen and Griffiths, 1973). In a rat seizure model, GGT activity was increased after 5 consecutive daily electroshock deliveries (Erakovicet al., 2001).
Decreases in various peripheral amino acids are associated with KD-mediated seizure suppression in animals and humans (Sariego-Jamardo et al., 2015). Previous studies also highlight KD-induced increases in brain GABA in animal models (Caldero ́n et al., 2017) and in humans (Dahlin et al., 2005; Wang et al., 2003). Future research is needed to determine whether peripheral amino acid restriction alters brain GABA/ glutamate metabolism.
The gut microbiota can impact levels of various neuroactive molecules in the periphery and in the brain itself (Vuong et al., 2017). We find that diet- and microbiota-dependent seizure pro- tection is associated with elevations in bulk GABA relative to glutamate content in the hippocampus (Figure 6F). Future studies are needed to determine whether other brain regions are similarly affected and whether GABA localized particularly to neuronal synapses or intracellular vesicles are also modulated by the gut microbiota. Consistent with a role for the gut micro- biota in modulating brain GABA levels, a previous study reveals that dietary fermentation by the gut microbiota modulates GABA levels in hypothalamic extracts (Frost et al., 2014). In addition, chronic Lactobacillus treatment elevates GABA levels in hippo- campal and prefrontal cortex as detected by magnetic reso- nance spectroscopy (Janik et al., 2016). While our study exam- ines microbial and metabolic mechanisms underlying how the gut microbiota influences seizure outcomes, further interroga- tion of the precise neurological mechanisms underlying the anti-seizure effects of the KD and KD-associated microbiota is needed. Of particular interest is the question of whether the gut microbiota modulates seizure susceptibility and incidence via alterations in excitatory/inhibitory balance and neurotrans- mission in particular neural circuits.
Overall, our study reveals a novel role for the gut microbiota in mediating and conferring seizure protection in two mouse models for refractory epilepsy. While the results lend credence to future research examining the gut microbiota in human epi- lepsy, several additional studies are needed to determine whether microbe-based treatments can be safely and effectively applied for clinical amelioration of seizure severity and incidence.
Cell 173, 1–14, June 14, 2018 a 2018 Elsevier Inc. 1