Abstracts

Rationale: Interictal hypometabolism is an important feature of many epileptic syndromes but has not been reported in Dravet syndrome (DS), a catastrophic childhood epilepsy associated with mutations in a voltage-activated sodium channel, Nav1.1 (SCN1A). Seizures in these children are often pharmacoresistant but can respond favorably to metabolic therapies such as the ketogenic diet. This raises the interesting possibility that metabolic dysfunction may occur in DS and contribute to its underlying pathophysiology.Methods: We developed novel methodology to assess in real-time changes in bioenergetics in zebrafish larvae between 4 and 6 days post fertilization (dpf). The scn1lab mutant zebrafish is the first simple vertebrate model that recapitulates many of the features of DS (Nat Commun, 2013, 4:2410). Glycolytic flux and mitochondrial respiration were simultaneously assessed in wildtype and scn1lab mutant zebrafish using a Seahorse Biosciences extracellular flux analyzer (XF analyzer). Neuronal excitability was increased by treatment with a classical potassium channel blockers, 4-aminopyridine (4-AP).Results: Scn1Lab mutant zebrafish showed a 1.7-fold decrease in baseline glycolytic rate and a 1.5-fold lower baseline oxygen consumption rate (OCR) compared to controls (n = 15, p < 0.05). Increasing neuronal excitability with the chemoconvulsant 4-AP resulted in an immediate increase in glycolytic rates in wild-type zebrafish; whereas mitochondrial OCR increased slightly and quickly recovered to baseline values. In contrast, scn1Lab mutant zebrafish showed a significantly slower and exaggerated increase of both glycolytic rates and OCR after 4-AP stimulation. A glucose metabolism PCR array identified five genes that were down-regulated in scn1Lab mutant zebrafish. Treatment of scn1lab mutant zebrafish with a version of the ketogenic diet for 2 days has been previously shown to reduce seizures. Here we show that KD increases both glycolysis and mitochondrial respiration rates in scn1lab mutant zebrafish to wt levels (n = 5). In addition, 4-AP stimulation induces a faster and dampened increase in both glycolysis and mitochondrial respiration in KD treated mutant zebrafish compared to vehicle treated mutant zebrafish. In summary, the KD returns mutant zebrafish metabolism to wildtype levels.Conclusions: Our results suggest a metabolic impairment concomitant with a sodium mutation in DS. We propose the metabolic impairment is due to changes in gene expression related to glucose metabolism. Increasing neuronal excitability with 4-AP also increases expression of genes regulating glucose metabolism and, in turn, glycolytic and oxidative phosphorylation rates. Taken together, our results suggest improving metabolic function may improve the seizure phenotype, as noted with the success of the ketogenic diet in these zebrafish and some patients. At the very least, it suggests that antiepileptic therapy that also affects metabolism should be carefully monitored when used in DS patients. (NIH R01NS39587(MP) and R01NS079214 (SCB))