Altered Glycolysis and Mitochondrial Respiration in a Zebrafish Model of Dravet Syndrome

Abstract

Altered metabolism 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). To address this, we developed novel methodology to assess real-time changes in bioenergetics in zebrafish larvae between 4 and 6 d postfertilization (dpf). Baseline and 4-aminopyridine (4-AP) stimulated glycolytic flux and mitochondrial respiration were simultaneously assessed using a Seahorse Biosciences extracellular flux analyzer. Scn1Lab mutant zebrafish showed a decrease in baseline glycolytic rate and oxygen consumption rate (OCR) compared to controls. A ketogenic diet formulation rescued mutant zebrafish metabolism to control levels. Increasing neuronal excitability with 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. The underlying mechanism of decreased baseline OCR in scn1Lab mutants was not because of altered mitochondrial DNA content or dysfunction of enzymes in the electron transport chain or tricarboxylic acid cycle. Examination of glucose metabolism using a PCR array identified five glycolytic genes that were downregulated in scn1Lab mutant zebrafish. Our findings in scn1Lab mutant zebrafish suggest that glucose and mitochondrial hypometabolism contribute to the pathophysiology of DS.

Keywords: Dravet syndrome; epilepsy; glycolysis; metabolism; mitochondrial respiration; zebrafish.

Figure 1.

Glycolytic and mitochondrial respiration rates in WT and scn1Lab mutant zebrafish at baseline and after 4-AP stimulation. A, B, Scn1Lab mutant zebrafish have lower baseline glycolytic and mitochondrial respiration rates than WT zebrafish. 4-AP immediately increases glycolytic and mitochondrial respiration rates in WT zebrafish. Scn1Lab mutant zebrafish have a delayed response to 4-AP of ∼30 min. Statistical analysis shows changes relative to time-matched untreated controls; points represent means±S.E.M. N = 14 (WT), 16 (scn1Lab), 16 (WT+4-AP), 14 (scn1Lab+4-AP) individual animals, mean±S.E.M. A, WT vs scn1Lab: p < 0.0001^a; WT vs WT+4-AP, p = 2.38e-30 (8 min)^o, p = 8.41e-30 (16 min)^p, p = 3.50e-26 (24 min)^q, p = 3.99e-24 (32 min)^r, p = 2.92e-25 (40 min)^s, p = 1.70e-21 (48 min)^t; scn1Lab vs scn1Lab+4-AP: p = 3.28e-6 (8 min)^u, p = 1.65e-11 (16 min)^v, p = 1.86e-13 (24 min)^w, p = 3.08e-17 (32 min)^x, p = 2.34e-14 (40 min)^y, p = 3.52e-15 (48 min)^z. B, WT vs scn1La: p < 0.0001^aa; scn1Lab versus scn1Lab+4-AP: p = 0.00071 (32 min)^bb, p = 2.20e-5 (40 min)^cc, p = 7.24e-5 (48 min)^dd. C, D, Locomotion plots for behavioral seizure activity in WT zebrafish exposed to 4 mmm 4-AP. Bar plot showing the mean ± SEM for WT fish at baseline, 8 min after exposure to 4-AP and 48 min after exposure to 4-AP. N = 48 WT fish; Kruskal–Wallis one-way ANOVA on ranks with a post hoc Tukey test. p < 0.05^c,d (8 min vs baseline).

Figure 2.

Respiratory chain complex activity in WT and scn1Lab mutant zebrafish. A, ECAR and OCR from Figure 1 are replotted to demonstrate the metabolic field and the increases in metabolic field after treatment with 4-AP. Scn1Lab mutant zebrafish increase metabolism to approach the metabolic state of WT zebrafish after 4-AP, suggesting mutant zebrafish retain a similar metabolic capacity as WT zebrafish; each point represents mean±S.E.M. B, Relative mitochondrial copy number was determined by total mitochondrial DNA. No significant differences were found (n = 3 individual animals per group; one-way ANOVA, p = 0.2056^gg). C, There is no difference in activity in complexes I–IV in WT and scn1Lab mutant zebrafish. Bars represent the mean ± SEM relative to WT activity, n = 3 groups with 25–30 fish pooled per group. Two-way ANOVA, interaction, p = 0.9367^hh. D, There are no differences in activity in selected TCA cycle enzymes in WT and scn1Lab mutant zebrafish. Bars represent the mean ± SEM relative to WT activity, n = 4 groups with 30 fish per group. Two-way ANOVA, interaction, p = 0.5801ⁱⁱ.

Figure 3.

Glucose metabolism-related gene expression in WT and scn1Lab mutant zebrafish (DRVT) at baseline and after 4-AP stimulation. A, Heatmap demonstrating relative expression of all genes analyzed from array. B, Schematic depicting the pathways in which up or downregulated genes are involved. Red color indicates downregulated genes in scn1Lab mutant zebrafish vs WT at baseline. C, Graph showing the seven genes with twofold or greater changes relative to respective controls. n = 6 pooled embryos per group analyzed once. Inset, PCR verification of genes for which specific primers were available, pck2, pck4, and pdk2. N = 3 groups of pooled embryos (6 per group) analyzed in triplicate, mean±S.E.M.

Figure 4.

Ketogenic diet restores metabolism of scn1Lab mutant zebrafish to WT levels. A, Metabolic profile of WT zebrafish is shifted slightly to be more glycolytic after KD treatment. Mutant zebrafish increase both glycolysis and mitochondrial respiration to WT levels. B, KD treatment reduces mutant zebrafish response to 4-AP to similar to WT levels. N = 4 (WT), 4 (scn1Lab), 5 (WT+KD), 5 (scn1Lab+KD), p = .017^jj. Values or bars indicate mean±S.E.M.

Figure 5.

Summary diagram depicting proposed mechanism. Mechanism demonstrating changes in glycolysis and mitochondrial respiration in scn1 mutant zebrafish at baseline (in black) and proposed action of KD (in red).

Figure 6.

Metabolic differences in WT and scn1lab mutant zebrafish are reproducible when grown in a separate facility, as well as with different instrumentation. All zebrafish in this figure were bred and grown at UCD. Using the same instrumentation as Figures 1 and 4 (XF24) the baseline differences are recapitulated in zebrafish grown at UCSF. The newer model of extracellular flux analysis (XF24e) is more sensitive and thus has higher baseline values for both glycolysis and mitochondrial respiration. A, B, Absolute baseline differences in glycolysis and respiration are recapitulated in both the XF24 and newer XF24e. A, XF24, p < .000^kk; XF24e, p < .00^ll; B, XF24, p = .00^mm; XF24e, p < .00ⁿⁿ. C, D, Although the XF24e is more sensitive, the relative differences in glycolysis and respiration are similar in both the XF24 and XF24e. C, XF24, p < .00^oo; XF24e, p < .00^pp. D, XF24, p = .0002^qq; XF24e, p < .0001^rr. Bars represent mean±S.E.M.