Glut1 deficiency (G1D): epilepsy and metabolic dysfunction in a mouse model of the most common human phenotype

Abstract

Brain glucose supplies most of the carbon required for acetyl-coenzyme A (acetyl-CoA) generation (an important step for myelin synthesis) and for neurotransmitter production via further metabolism of acetyl-CoA in the tricarboxylic acid (TCA) cycle. However, it is not known whether reduced brain glucose transporter type I (GLUT-1) activity, the hallmark of the GLUT-1 deficiency (G1D) syndrome, leads to acetyl-CoA, TCA or neurotransmitter depletion. This question is relevant because, in its most common form in man, G1D is associated with cerebral hypomyelination (manifested as microcephaly) and epilepsy, suggestive of acetyl-CoA depletion and neurotransmitter dysfunction, respectively. Yet, brain metabolism in G1D remains underexplored both theoretically and experimentally, partly because computational models of limited brain glucose transport are subordinate to metabolic assumptions and partly because current hemizygous G1D mouse models manifest a mild phenotype not easily amenable to investigation. In contrast, adult antisense G1D mice replicate the human phenotype of spontaneous epilepsy associated with robust thalamocortical electrical oscillations. Additionally, and in consonance with human metabolic imaging observations, thalamus and cerebral cortex display the lowest GLUT-1 expression and glucose uptake in the mutant mouse. This depletion of brain glucose is associated with diminished plasma fatty acids and elevated ketone body levels, and with decreased brain acetyl-CoA and fatty acid contents, consistent with brain ketone body consumption and with stimulation of brain beta-oxidation and/or diminished cerebral lipid synthesis. In contrast with other epilepsies, astrocyte glutamine synthetase expression, cerebral TCA cycle intermediates, amino acid and amine neurotransmitter contents are also intact in G1D. The data suggest that the TCA cycle is preserved in G1D because reduced glycolysis and acetyl-CoA formation can be balanced by enhanced ketone body utilization. These results are incompatible with global cerebral energy failure or with neurotransmitter depletion as responsible for epilepsy in G1D and point to an unknown mechanism by which glycolysis critically regulates cortical excitability.

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

FIGURE 1. Brain GLUT-1 expression

(A) GLUT-1 expression was determined by western blot in forebrain, cortex, thalamus and striatum of adult transgenic (n=4) and control mice (n=4). The two GLUT-1 isoforms (55 kDa GLUT-1 in endothelial cells and 45 kDa GLUT-1 in astrocytes) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are illustrated. (B) GLUT-1 and GAPDH signals were quantified using an optical densitometer. The densities of GLUT-1 signal were normalized to GAPDH signal. The averaged GLUT-1/GAPDH ratios for forebrain, cortex, thalamus and striatum are illustrated (mean ± SEM). In forebrain, cortex and thalamus, the relative GLUT-1 expression is significantly reduced in G1D mice relative to controls. In the striatum, however, there were no statistical differences in GLUT-1 expression between the animal groups. * P <0.05, ** P<0.01.

FIGURE 2. Brain 14C-deoxyglucose accumulation determined by autoradiography

The upper panel shows a schematic representation of 14C-2-deoxyglucose distribution in normal brain by autoradiography. The averaged ratio (with SEM in brackets) of deoxyglucose accumulation in cortex (marked as 1 in the upper panel), striatum (2 in the upper panel) and thalamus (3 in the upper panel) between G1D and control animals is illustrated in the lower panel. All accumulation values were significantly lower in G1D brain regions relative to normal mice. In agreement with FDG-PET data, deoxyglucose distribution was notably lower in the cortex and thalamus of the G1D compared to the control animal. However, accumulation in the striatum was slightly lower in G1D, which is consistent with the similar expression of GLUT-1 in this brain region across both animal groups.

FIGURE 3. Representative EEG activity recorded from awake G1D mice

The top EEG Trace shows a typical epileptic spike seen in all G1D mice. The bottom EEG Trace shows a series of spike and spike-wave discharges associated with epileptiform activity within the left frontal cortex. The spectral power of the EEG signal is represented by the bottom plot which shows an increase of spectral power in the theta frequency band (4–8 Hz) during the indicated spike-wave discharge shown above (bracketed arrow to white ellipse) and also in three subsequent events (white ellipses) which occurred during this representative one hour window of EEG recording. Left Axis: normalized power with representative graybar intensity, Bottom Axis: time in minutes, Right Axis: frequency in Hz.

FIGURE 4. Thalamocortical oscillations in G1D brain slice

Left panel: Thalamocortical slice preparation with 64-channel multi electrode array (MEA) positioned over the sensory barrel cortex. Center panel: Normal mouse: Field potential recording from MEA array positioned as in left panel showing the 16 electrode positions as designated by the white box outlined in left panel. Right panel: G1D mouse: note the ~3 Hz oscillation in all the electrodes indicative of thalamocortical hypersynchronization. Slices were bathed in drug-free, 2.5 mM glucose-containing perfusate.

FIGURE 5. Astrocytic proliferation with preservation of glutamine synthase (GS) in G1D

Z-stacked, 10×, 40 μm confocal images of the barrel cerebral cortex (4 normal and 5 G1D entire brains were studied at 240 μm coronal section intervals) at P28 (A) and P120 (B) after reaction with antibodies anti-glial fibrillary acidic protein (GFAP) and anti-GS. G1D is associated with astrocytic proliferation, which is more prominent in adult animal but, importantly, not with GS depletion.

FIGURE 6. Cerebral free fatty acids, cholesterol and triglycerides in G1D

Lipids were measured in brain extracts of G1D and control (C) mice (n=6, age: 4–5 months) in the fed state. Lower concentration of fatty acids in brain of G1D mice relative to controls was identified, whereas cholesterol (total and free) and triglycerides were equally abundant in both groups.