Research ArticlePYRUVATE DEHYDROGENASE DEFICIENCY

Brain metabolism modulates neuronal excitability in a mouse model of pyruvate dehydrogenase deficiency

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Science Translational Medicine  20 Feb 2019:
Vol. 11, Issue 480, eaan0457
DOI: 10.1126/scitranslmed.aan0457
  • Fig. 1 Characterization of the PDHD mouse.

    (A) Genotyping of the Pdha1 locus in brain (B), liver (L), heart (H), and skeletal muscle (M) of male offspring produced from mating a Pdha1flox8/flox8 female with an hGFAP-Cre male. Top gel: Genotyping of wild-type (WT; 700 bp), floxed (800 bp), and null (400 bp) allele of Pdha1. Bottom gel: PCR of Cre transgene (350 bp). +, +/−, and − represent Pdha1flox8/flox8, Pdha1flox8/wt, and Pdha1wt/wt, respectively. (B) Qualitative and quantitative expression of Pdha1 in cortex of PDHD and control males by Western blotting (n = 4 mice per group). (C) Active and total PDH enzymatic activity in brain of PDHD and control males (n = 8 mice per group). (D) Concentration of metabolites in forebrain from both mouse groups (n = 6 mice per group) determined by gas chromatography–mass spectrometry (GC-MS). Data represent means ± SEM. Statistical differences were determined using two-tailed Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 2 Metabolism of [U-13C]glucose in the cerebral cortex.

    (A) 13C NMR spectrum of control and PDHD mouse cortex (control, n = 7; PDHD, n = 5). For comparison, the height of taurine C1 spectrum was equalized in both spectra because taurine is not enriched by 13C-glucose and its concentration was not different between groups (control, 16.35 ± 0.51 μmol/g of brain; PDHD, 14.16 ± 1.06 μmol/g of brain). (B) 13C enrichment and concentration of metabolites in control and PDHD cortex estimated by 1H NMR spectroscopy in all the samples in (A) (control, n = 7; PDHD, n = 5). (C) Isotopomer analysis of glutamate C4, glutamine C4, and GABA C2 in PDHD and control cortex using the spectra from all the samples in (A) (control, n = 7; PDHD, n = 5). GLU, glutamate; GLN, glutamine; ASP, aspartate; ALA, alanine; NAA, N-acetylaspartate; LAC, lactate; Ac-CoA, acetyl-CoA. C#, carbon labeled in position. These abbreviations also apply to Fig. 3. Data indicate mean ± SEM. Differences were determined by a two-tailed Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 3 Metabolism of [1,6-13C]glucose and [1,2-13C]acetate in the cerebral cortex.

    (A) Representative 13C NMR spectra of control and PDHD mouse cortex (n = 10). The blue and red bands represent the expanded regions shown (D). For illustration purposes, the amplitude of taurine C1 was equated in both spectra because taurine is not enriched by neither 13C-acetate ([1,2-13C]acet) nor 13C-glucose ([1,6-13C]gluc), and its concentration was not different between groups (control, 15.3 ± 0.5 μmol/g of brain; PDHD, 16.4 ± 0.6 μmol/g of brain). (B) 13C metabolite enrichment and concentration in cortex from PDHD and control (n = 10 in each group). (C) 13C NMR spectra and isotopomer analysis of glutamate C4 and glutamine C4 in PDHD and control. (D) Acetate:glucose oxidative ratio in cortex from PDHD and control mice. Values indicate means ± SEM. Differences were determined with a two-tailed Student’s t test. **P < 0.01 and ***P < 0.001.

  • Fig. 4 Spontaneous cortical electrical activity in human and mouse PDHD.

    (A) Top: Location of human EEG electrodes. Middle and bottom: Recordings (band-pass filtered 20 to 50 Hz) from a 16-week-old healthy infant control (control human EEG) and a 10-week-old patient with PDHD (PDHD patient EEG). Scale bar, 50 ms/10 μV. (B) Human EEG power spectra and coherence in patients with PDHD (patients >5 months old are depicted in fig. S5B). Black arrows indicate peaks in EEG power spectra in a control subject. Roman numerals refer to human subject indexed in fig. S5A. Inset: Periodicity of epochs/bursts of mid- and high-frequency oscillations in a control (blue) and in patients with PDHD (additional details are in fig. S7, A to C). (C) Top: In vivo spontaneous local field potential (LFP) recorded in the mouse somatosensory cortex. Middle and bottom: Example recordings (band-pass filtered 10 to 50 Hz). Scale bar, 25 ms/200 μV. (D) Top: Average LFP power (thick line) in PDHD (n = 13 sites, 13 mice) compared to littermate controls (n = 10 sites, 10 mice). Flanking thinner lines reflect SEM. Bottom: Average power spectra within each frequency band. The lower-upper limits of each frequency band are shown in the x axis of the power-frequency graph above. (E) Coherence between recordings in layers 2/3 and 4/5 in PDHD (n = 13 sites, 13 mice) compared to control (n = 10 sites, 10 mice). Data indicate means ± SEM. Statistical differences were determined using two-tailed Student’s t test and Mann-Whitney U test. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 5 Local evoked response and paroxysmal epileptiform activity in PDHD mouse cortex.

    (A) LFP evoked in layer 2/3 by local microstimulation 300 μm below the recording electrode. (B) Amplitudes of LFPs evoked in layer 2 after stimulation in layer 4 for a range of currents (input/output curve) in PDHD (n = 6 recording sites, 3 mice) and control (n = 7 sites, 6 mice). (C) Paired-pulse ratios [LFP evoked for second to sixth stimulus/LFP evoked for the first stimulus in a train (mean LFP2 to 6/LFP1)] in PDHD (n = 8 sites, four mice) and control (n = 7 sites, four mice). (D) Recordings from layer 2/3 barrel cortex illustrating paroxysmal epileptiform bursts of action potentials in PDHD (PDHD, 7 sites and 7 mice; control, 13 sites and 13 mice). Left: Example multiunit action potential recording traces from control and PDHD cortex (trace within dotted box is expanded below). Middle and right: Group average of multiunit spike firing rate and number of multiunit spike bursts greater than 3 s. (E) EEG recording from a subdural electrode placed over the barrel cortex of an awake PDHD mouse. Raw EEG traces (top) and spectrogram (bottom) depicting paroxysmal epileptiform activity. dB, decibels. Data indicate means ± SEM. Statistical differences were determined using two-tailed Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 6 Whisker pad–evoked single-unit firing and associated inhibition in PDHD mice.

    (A) Left: Experimental design and example single-unit waveforms. The average waveform (darker color) is superimposed on 100 waveforms (lighter color). Scale bar, 500 μs/0.1 mV. Right: Example dot-raster plots depicting action potentials induced by whisker pad stimulation with the PSTH below. (B) Single-unit group data from control (n = 13 units, five mice) and PDHD (n = 10, four mice) illustrating jitter, peak latency, end latency, and duration of action potentials. (C) Left: Waveforms of broad regular spiking (putative excitatory) and narrow spiking (putative fast-spiking inhibitory) indicating the parameters (trough-to-peak time and half-widths) used to classify spikes into RS or FS. Right: Bar charts show RS (n = 11, black) and FS (n = 7, green) single units from PDHD mice normalized to the mean of the corresponding group in control. (D) RS and FS recorded simultaneously from the same electrode (schematic on acetate:glucose) in PDHD mice (n = 10 RS to FS pairs) and control (n = 4 RS to FS pairs). (E) Left: Diagram of narrow-tip (high-resistance) whole-cell patch clamp recording and traces obtained in response to a 125-pA step current in control (blue) and PDHD (red) neurons. Right: Population average for a number of action potentials elicited by current steps in regular-spiking (control, n = 9 cells, 7 mice; PDHD, n = 17 cells, 13 mice) and fast-spiking (control, n = 22 cells, 12 mice, PDHD n = 26 cells, 15 mice) cells from PDHD and control mice. (F) Population average input resistance in PDHD (n = 14 cells, 12 mice) and in control fast-spiking neurons (n = 8 cells, 7 mice) and regular-spiking neurons (PDHD, n = 16 cells, 10 mice; controls, n = 13 cells, 8 mice). Data indicate means ± SEM. Statistical differences were determined using two-tailed Student’s t test and two-way analysis of variance (ANOVA) with correction for multiple comparisons by controlling the false discovery rate with the Bengamini-Krieger-Yekutieli method. *P < 0.05, **P < 0.01, and ****P < 0.0001.

  • Fig. 7 Machine learning algorithm using a support vector for ictal prediction in PDHD mice.

    (A) Outline of a machine learning algorithm with 10-fold cross-validation for ictal prediction. (B) Left: Two-dimensional (2D) example scatter plot of normalized (norm.) gamma and beta power for mouse preictal events contrasted with power of interictal EEG segments over 2 hours. Right: 3D scatter plot of the same data. (C) Receiver operating characteristic curve of analysis on data from (B). (D) Classifier performance measures for group data (n = 5 PDHD mice).

  • Fig. 8 Acetate enhancement of cortical activation and glutamate 13C labeling in PDHD mice.

    (A) Top: Spontaneous LFP recordings from a low (delta frequency) and higher frequency bin (low gamma) before and 1 hour (hr) after an intraperitoneal (ip) injection (2 mg/g of body weight) of sodium acetate. Bottom: LFP power after acetate in a PDHD mouse. Inset: Temporal profile of gamma power after acetate administration (injected at 10 min). (B and C) LFP power and coherence mean percentage change (compared to pre-acetate baseline) for all mice (PDHD, n = 5; control, n = 5) after acetate (black arrow). Black lines in (C) and (D) enclose statistically significant regions (P < 0.05). (D) Representative spectra of glutamate C4 (glut. C4) and GABA C2 from PDHD and control mice. The contribution of 13C-acetate (13C-acet) [(area D45 + area Q)/total area] and 13C-glucose (13C-gluc)] [(area S + area D34)/total area] was calculated for both isotopomers. The graphs immediately below represent the contribution of acetate and glucose to glutamate and GABA in control (top) and PDHD mice (middle). The bottom graph depicts the contribution to glutamate:GABA ratio from acetate and glucose in control and PDHD mice. Data represent mean ± SEM. Statistical differences were determined using two-tailed Student’s t test. *P < 0.05 and ***P < 0.001.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/480/eaan0457/DC1

    Methods

    Fig. S1. Glucose metabolism schema and survival curves of patients and mice with PDHD.

    Fig. S2. Expression of PDH in the mouse somatosensory cortex.

    Fig. S3. Phenotype of PDHD mice.

    Fig. S4. Glucose metabolism in PDHD mice.

    Fig. S5. EEG in patients with PDHD.

    Fig. S6. Patient with PDHD EEG (patient I).

    Fig. S7. EEG oscillation epochs in patients with PDHD.

    Fig. S8. Cortical layering, evoked LFP, and baseline EEG in PDHD mice.

    Fig. S9. Sorting of multiunit action potentials into single units and separation into broad- and fast-spiking neuronal origin.

    Fig. S10. In vivo RS neuron discharge relative to fast-spiking activity.

    Fig. S11. Brain slice intracellular recordings illustrating cell discrimination, rheobase, and firing.

    Fig. S12. Neuronal input resistance under energetic substrate deprivation.

    Fig. S13. EEG gamma power in relation to epileptiform events in PDHD.

    Fig. S14. Gamma power in relation to epileptiform events in additional patients with PDHD.

    Fig. S15. Acetate modulation of evoked LFPs in vivo.

    Fig. S16. Acetate modulation of spontaneous excitatory postsynaptic currents.

    Fig. S17. Modulation of input resistance and firing with acetate.

    Fig. S18. Acetate modulation of EEG epileptiform events in awake mice.

    Fig. S19. EEG epileptiform events in awake mice with vehicle injections.

    Fig. S20. Representation of glucose oxidation and excitability coupling in relation to PDH activity.

    Table S1. Plasma metabolite concentration.

    Table S2. Isotopomer analysis of [U-13C]glucose NMR spectra from cerebral cortex NMR spectra.

    Table S3. Isotopomers derived from [1,2-13C]acetate and [1,6-13C]glucose in cerebral cortex NMR spectra.

    Table S4. Metabolic model analysis of glutamate and glutamine C4 multiplets from [1,6-13C]glucose and [1,2-13C]acetate cerebral cortex spectra.

    Table S5. Modified Racine’s scale.

    Table S6. Key clinical features of patients with PDHD.

    Table S7. Raw data (provided as a separate Excel file).

    Movie S1. PDHD mouse video-EEG recording of epileptiform events.

    References (5775)

  • The PDF file includes:

    • Methods
    • Fig. S1. Glucose metabolism schema and survival curves of patients and mice with PDHD.
    • Fig. S2. Expression of PDH in the mouse somatosensory cortex.
    • Fig. S3. Phenotype of PDHD mice.
    • Fig. S4. Glucose metabolism in PDHD mice.
    • Fig. S5. EEG in patients with PDHD.
    • Fig. S6. Patient with PDHD EEG (patient I).
    • Fig. S7. EEG oscillation epochs in patients with PDHD.
    • Fig. S8. Cortical layering, evoked LFP, and baseline EEG in PDHD mice.
    • Fig. S9. Sorting of multiunit action potentials into single units and separation into broad- and fast-spiking neuronal origin.
    • Fig. S10. In vivo RS neuron discharge relative to fast-spiking activity.
    • Fig. S11. Brain slice intracellular recordings illustrating cell discrimination, rheobase, and firing.
    • Fig. S12. Neuronal input resistance under energetic substrate deprivation.
    • Fig. S13. EEG gamma power in relation to epileptiform events in PDHD.
    • Fig. S14. Gamma power in relation to epileptiform events in additional patients with PDHD.
    • Fig. S15. Acetate modulation of evoked LFPs in vivo.
    • Fig. S16. Acetate modulation of spontaneous excitatory postsynaptic currents.
    • Fig. S17. Modulation of input resistance and firing with acetate.
    • Fig. S18. Acetate modulation of EEG epileptiform events in awake mice.
    • Fig. S19. EEG epileptiform events in awake mice with vehicle injections.
    • Fig. S20. Representation of glucose oxidation and excitability coupling in relation to PDH activity.
    • Table S1. Plasma metabolite concentration.
    • Table S2. Isotopomer analysis of [U-13C]glucose NMR spectra from cerebral cortex NMR spectra.
    • Table S3. Isotopomers derived from [1,2-13C]acetate and [1,6-13C]glucose in cerebral cortex NMR spectra.
    • Table S4. Metabolic model analysis of glutamate and glutamine C4 multiplets from [1,6-13C]glucose and [1,2-13C]acetate cerebral cortex spectra.
    • Table S5. Modified Racine’s scale.
    • Table S6. Key clinical features of patients with PDHD.
    • Legend for table S7
    • Legend for movie S1
    • References (5775)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S7. Raw data (provided as a separate Excel file).
    • Movie S1 (.mp4 format). PDHD mouse video-EEG recording of epileptiform events.

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