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Research ArticleNew Research, Disorders of the Nervous System

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

Maneesh G. Kumar, Shane Rowley, Ruth Fulton, Matthew T. Dinday, Scott C. Baraban and Manisha Patel
eNeuro 29 March 2016, 3 (2) ENEURO.0008-16.2016; DOI: https://doi.org/10.1523/ENEURO.0008-16.2016
Maneesh G. Kumar
1Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045
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Shane Rowley
1Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045
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Ruth Fulton
1Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045
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Matthew T. Dinday
2Department of Neurological Surgery, University of California San Francisco, San Francisco, California 94143
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Scott C. Baraban
2Department of Neurological Surgery, University of California San Francisco, San Francisco, California 94143
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Manisha Patel
1Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045
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  • Figure 1.
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    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 < 0.0001a; 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 < 0.0001aa; 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 < 0.05c,d (8 min vs baseline).

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    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 = 0.2056gg). 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 = 0.9367hh. 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.

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    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.

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    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 = .017jj. Values or bars indicate mean±S.E.M.

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    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).

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    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 < .000kk; XF24e, p < .00ll; B, XF24, p = .00mm; 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 < .00oo; XF24e, p < .00pp. D, XF24, p = .0002qq; XF24e, p < .0001rr. Bars represent mean±S.E.M.

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    Table 1.

    Statistical table

    Data structureType of testConfidence interval
    aNormal distributiont test−6.78 to −3.94
    bNormal distributiont test−116.0 to −98.31
    cNon-normal distribution (Shapiro–Wilk test p < 0.05)One-way ANOVA Kruskal–Wallis on ranks with a post hoc Tukey testp < 0.01 ANOVA
    8 min vs baseline: p < 0.05
    dNon-normal distribution (Shapiro–Wilk test p < 0.05)One-way ANOVABaseline vs 48 min: p > 0.05
    eNormal distributionUnpaired t test−23.76 to −15.36
    fNormal distributiont test with Holm–Sidak for multiple comparisons−245.2 to −110.5
    gNormal distributiont test with Holm–Sidak for multiple comparisons−64.78 to −20.95
    hNormal distributionOne-way ANOVAWT vs WT+4-AP−0.9128 to 0.1422
    WT vs scn1Lab−0.5763 to 0.4787
    WT vs scn1Lab+4-AP−0.5664 to 0.4886
    iNormal distributionTwo-way ANOVAComplex I−148.8 to 112.6
    Complex II−138.4 to 123.1
    Complex III−177.9 to 83.56
    Complex IV−146.6 to 114.9
    jNormal distributionTwo-way ANOVAAconitase−15.04 to 45.86
    Fumarase−32.52 to 28.38
    Mal. Dehyd.−21.06 to 33.41
    kNormal distributiont test−0.3722 to 3.172
    lNormal distributiont test65.33 to 64.27
    mNormal distributiont test−236.8 to 132.0
    nNormal distributiont test−1.676 to 0.3118
    oNormal distributiont test with Holm–Sidak for multiple comparisons−33.97 to −22.24
    pNormal distributiont test with Holm–Sidak for multiple comparisons−33.59 to −21.85
    qNormal distributiont test with Holm–Sidak for multiple comparisons−31.04 to −19.30
    rNormal distributiont test with Holm–Sidak for multiple comparisons−29.59 to −17.86
    sNormal distributiont test with Holm–Sidak for multiple comparisons−30.39 to −18.66
    tNormal distributiont test with Holm–Sidak for multiple comparisons−27.73 to −16.00
    uNormal distributiont test with Holm–Sidak for multiple comparisons−18.01 to −6.278
    vNormal distributiont test with Holm–Sidak for multiple comparisons−24.10 to −12.37
    wNormal distributiont test with Holm–Sidak for multiple comparisons−26.07 to −14.33
    xNormal distributiont test with Holm–Sidak for multiple comparisons−29.68 to −17.95
    yNormal distributiont test with Holm–Sidak for multiple comparisons−26.95 to −15.21
    zNormal distributiont test with Holm–Sidak for multiple comparisons−27.74 to −16.01
    aaNormal distributiont test−116.0 to −98.31
    bbNormal distributiont test with Holm–Sidak for multiple comparisons−112.2 to −6.900
    ccNormal distributiont test with Holm–Sidak for multiple comparisons−128.0 to −22.77
    ddNormal distributiont test with Holm–Sidak for multiple comparisons−122.9 to −17.64
    eeNormal distributionOne-way ANOVAControl vs 10 μm−3.649 to 7.982
    Control vs 50 μm−6.585 to 5.046
    Control vs 100 μm−4.812 to 6.819
    Control vs 1 mm−6.324 to 5.307
    Control vs 4 mM−7.943 to 2.825
    ffNormal distributionOne-way ANOVAControl vs 10 μm−67.77 to 93.79
    Control vs 50 μm−62.06 to 99.49
    Control vs 100 μm−39.08 to 122.5
    Control vs 1 mm−19.22 to 142.3
    Control vs 4 mm−19.85 to 129.7
    ggNormal distributionOne-way ANOVAWT vs WT+4-AP−0.9128 to 0.1422
    WT vs scn1Lab−0.5763 to 0.4787
    WT vs scn1Lab+4-AP−0.5664 to 0.4886
    hhNormal distributionTwo-way ANOVAComplex I−148.8 to 112.6
    Complex II−138.4 to 123.1
    Complex III−177.9 to 83.56
    Complex IV−146.6 to 114.9
    iiNormal distributionTwo-way ANOVAAconitase−15.04 to 45.86
    Fumarase−32.52 to 28.38
    Mal. Dehyd.−21.06 to 33.41
    jjNormal distributiont test1.783 to 8.531
    kkNormal distributiont test−4.128 to −2.727
    llNormal distributiont test−36.51 to −22.87
    mmNormal distributiont test−98.59 to −32.08
    nnNormal distributiont test−104.4 to −49.14
    ooNormal distributiont test−46.07 to −30.43
    ppNormal distributiont test−52.57 to −32.94
    qqNormal distributiont test−35.93 to −11.69
    rrNormal distributiont test−32.95 to −15.50
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Altered Glycolysis and Mitochondrial Respiration in a Zebrafish Model of Dravet Syndrome
Maneesh G. Kumar, Shane Rowley, Ruth Fulton, Matthew T. Dinday, Scott C. Baraban, Manisha Patel
eNeuro 29 March 2016, 3 (2) ENEURO.0008-16.2016; DOI: 10.1523/ENEURO.0008-16.2016

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Altered Glycolysis and Mitochondrial Respiration in a Zebrafish Model of Dravet Syndrome
Maneesh G. Kumar, Shane Rowley, Ruth Fulton, Matthew T. Dinday, Scott C. Baraban, Manisha Patel
eNeuro 29 March 2016, 3 (2) ENEURO.0008-16.2016; DOI: 10.1523/ENEURO.0008-16.2016
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Keywords

  • Dravet syndrome
  • epilepsy
  • Glycolysis
  • metabolism
  • Mitochondrial Respiration
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