BAD-dependent regulation of fuel metabolism and K(ATP) channel activity confers resistance to epileptic seizures

Abstract

Neuronal excitation can be substantially modulated by alterations in metabolism, as evident from the anticonvulsant effect of diets that reduce glucose utilization and promote ketone body metabolism. We provide genetic evidence that BAD, a protein with dual functions in apoptosis and glucose metabolism, imparts reciprocal effects on metabolism of glucose and ketone bodies in brain cells. These effects involve phosphoregulation of BAD and are independent of its apoptotic function. BAD modifications that reduce glucose metabolism produce a marked increase in the activity of metabolically sensitive K(ATP) channels in neurons, as well as resistance to behavioral and electrographic seizures in vivo. Seizure resistance is reversed by genetic ablation of the K(ATP) channel, implicating the BAD-K(ATP) axis in metabolic control of neuronal excitation and seizure responses.

Figure 1. Blunted Mitochondrial Metabolism of Glucose in Primary Bad^−/− and Bad ^S155A Neurons and Astrocytes

(A) Representative trace of oxygen consumption rate (OCR) measured in real time indicating basal and maximal respiration rates (BR and MR, respectively). (B and C) BR and MR in the presence of glucose in primary Bad^−/− cortical neurons (B) and astrocytes (C). (D and E) BR and MR in the presence of glucose in primary Bad ^S155A cortical neurons (D) and astrocytes (E). Data in B through E are presented as mean ± SEM; n=4–6 independent neuron or astrocyte cultures. * p < 0.05; **p < 0.01; *** p < 0.001; wild-type vs. Bad^−/−, or Bad ^S155A; two-tailed Student’s t-test. See also Figure S1.

Figure 2. Mitochondrial Metabolism of Non-Glucose Fuels in Bad^−/− and Bad ^S155A Neurons and Astrocytes

(A and B) Mitochondrial maximal respiration (MR) in wild-type and Bad^−/− neurons (A) and astrocytes (B) supplied with 5 mM L-glutamine or 5 mM L-lactate. (C and D) MR in the presence of 5 mM β-D-hydroxybutyrate in primary neurons (C) and astrocytes (D) derived from Bad^−/− and Bad ^S155A mice. Data in A through D are presented as mean ± SEM; n=4–6 independent neuron or astrocyte cultures. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s. in A and B, non-significant; wild-type vs. Bad^−/− or Bad ^S155A; two-tailed Student’s t-test

Figure 3. Resistance of Bad Genetic Models to Kainic Acid- and Pentylenetetrazole-Induced Acute Seizures

(A and B) Raw seizure scores in 8–10-wk old male Bad^−/− (A) and Bad ^S155A (B) mice compared with wild-type control animals over a 4-hour period after a single i.p. injection of kainic acid (KA) at 30 mg/kg. See also Figure S2. (C) Integrated seizure severity of the above experimental cohorts and Bid^−/− mice similarly treated with KA. The scoring scale and the formula used to derive seizure severity are provided. Wild-type, n=42; Bad^−/−, n=13; Bad ^S155A, n=20; Bid^−/−; n=9. (D) Integrated seizure severity in Bad^−/− and wild-type mice (n=16) subjected to a single s.c. injection of pentylenetetrazole (PTZ) at 80 mg/kg monitored over a 70 minute-period. See also Figures S3 and S4. Data in A through D are presented as mean ± SEM. *** p < 0.001; n.s., non-significant; two-tailed Student’s t-test.

Figure 4. EEGs of Kainic Acid-Induced Seizure Activity in Wild-Type and Bad^−/− Animals

(A) A single representative EEG channel recording of approximately 1 hour for each genotype (right frontoparietal electrode). A single i.p. injection of kainic acid (30 mg/kg of body weight) was administered at the indicated time. The green block arrow near the end of the wild-type recording indicates the time of death during generalized tonic-clonic status epilepticus. (B) The average fraction of time spent in a high energy spiking period for each genotype during a 30 minute period beginning 3 minutes after injection. EEGs were scored by an investigator blind to the genotype, guided by both the raw EEG traces and a trace of power in the 20–70 Hz band to identify onset and offset of high energy spiking (typically seen as rapid increases or decreases of ≥5 dB in power). Wild-type, n=17; Bad^−/−, n=20; mean ± SEM, * p < 0.05 by two-tailed Student’s t-test. In these cohorts, 9 of 17 wild-type and 0 of 20 Bad^−/− animals died during status epilepticus.

Figure 5. BAD Manipulations Increase K_ATP Channel Activity in Dentate Granule Neurons

(A) Continuous recordings of wild-type (left) or Bad^−/− (right) single-channel currents in cell-attached patches with zero applied voltage. Single-channel openings are seen as downward deflections of the current trace. (B) N·P_open values for WT and Bad^−/− single channel recordings. For the Bad^−/− recordings, the K_ATP blocker tolbutamide was added (Tolb; 200 μM) and then washed out (Washout). n=6 for each genotype. (C) Maximum activatable K_ATP conductance determined from the difference between the slope conductance at the time of break-in and after conductance run-up (G_max), with low ATP (0.3 mM) in the recording pipette, for the indicated genotypes and for WT in the presence of 200 μM tolbutamide (WT + Tolb). (D) “Washdown” of an initially high K_ATP conductance, with high ATP (4 mM) in the recording pipette, seen in Bad^−/− and Bad S155A but not in WT or in Bad^−/−; Kir6.2^−/−. The time course of slope conductance measured during whole-cell recording was normalized to the value 3 min after break-in. (E) “Washdown” (measured as in D) prevented by preincubation in 200 μM tolbutamide. Data in B through E are presented as mean ± SEM.

Figure 6. BAD Modulation of Seizure Resistance is Dependent on the K_ATP Channel

(A and B) Raw scores (A) and seizure severity (B) in Bad^−/− and Kir6.2^−/− single mutants and Bad^−/−; Kir6.2^−/− double mutant mice compared with wild-type mice after a single i.p. injection of kainic acid monitored and analyzed as in Figure 3. Data are presented as mean ± SEM. ** p < 0.01; *** p < 0.001; n.s., non-significant; two-tailed Student’s t-test.