Alteration of glial-neuronal metabolic interactions in a mouse model of Alexander disease

Abstract

Alexander disease is a rare and usually fatal neurological disorder characterized by the abundant presence of protein aggregates in astrocytes. Most cases result from dominant missense de novo mutations in the gene encoding glial fibrillary acidic protein (GFAP), but how these mutations lead to aggregate formation and compromise function is not known. A transgenic mouse line (Tg73.7) over-expressing human GFAP produces astrocytic aggregates indistinguishable from those seen in the human disease, making them a model of this disorder. To investigate possible metabolic changes associated with Alexander disease Tg73.7 mice and controls were injected simultaneously with [1-(13)C]glucose to analyze neuronal metabolism and [1,2-(13)C]acetate to monitor astrocytic metabolism. Brain extracts were analyzed by (1)H magnetic resonance spectroscopy (MRS) to quantify amounts of several key metabolites, and by (13)C MRS to analyze amino acid neurotransmitter metabolism. In the cerebral cortex, reduced utilization of [1,2-(13)C]acetate was observed for synthesis of glutamine, glutamate, and GABA, and the concentration of the marker for neuronal mitochondrial metabolism, N-acetylaspartate (NAA) was decreased. This indicates impaired astrocytic and neuronal metabolism and decreased transfer of glutamine from astrocytes to neurons compared with control mice. In the cerebellum, glutamine and GABA content and labeling from [1-(13)C]glucose were increased. Evidence for brain edema was found in the increased amount of water and of the osmoregulators myo-inositol and taurine. It can be concluded that astrocyte-neuronal interactions were altered differently in distinct regions.

Figure 1

Schematic presentation of labeling in glutamate, glutamine and GABA from [1-¹³C]glucose and [1,2-¹³C]acetate. Most of the [4-¹³C]glutamate observed is synthesized from [1-¹³C]glucose and located in glutamatergic neurons; it can also be generated from [4-¹³C]glutamine from astrocytes (A). Glutamatergic neurons release [4-¹³C]glutamate which can be converted to [4-¹³C]glutamine in astrocytes, which also can synthesize [4-¹³C]glutamate via their own TCA cycle (B). [1,2-¹³C]acetate is metabolized only in astrocytes and [4,5-¹³C]glutamine can be formed (C) which may be converted to [4,5-¹³C]glutamate in glutamatergic and GABAergic neurons (C). The latter can convert this glutamate to [1,2-¹³C]GABA (C). In GABAergic neurons [2-¹³C]GABA is formed from [1-¹³C]glucose (D). Interpretation of the results obtained from ¹³C MRS is based on the different metabolic fates of [1-¹³C]glucose and [1,2-¹³C]acetate (Figure 1). [1-¹³C]glucose enters both astrocytes and neurons approximately equally (Nehlig & Coles 2007) and is transformed via glycolysis to [3-¹³C]pyruvate (Figure 1). The latter can be transported into mitochondria to enter the TCA cycle as acetyl CoA or be converted to lactate, mostly in astrocytes, to be transported to neurons (Pellerin et al. 2007, Bak et al. 2007). In neurons lactate can be oxidized to pyruvate and then be converted to [2-¹³C]acetyl CoA and enter the TCA cycle. Approximately 70% of the acetyl CoA from glucose used for glutamate synthesis in the brain is metabolized in neurons and 30 % in astrocytes (Hassel et al. 1995, Qu et al. 2000). After several steps, [4-¹³C]glutamate is formed (Figure 1 A,B,D). In principal, some neuronal [4-¹³C]glutamate could be derived from [4-¹³C]glutamine synthesized in astrocytes from [1-¹³C]glucose (1B). However, Hassel et al. (1997) showed that [4-¹³C]glutamate formation was not impaired when astrocytic TCA cycle activity was stopped. In GABAergic neurons, [2-¹³C]GABA can be formed from [1-¹³C]glucose (Figure 1D). In astrocytes, [4-¹³C]glutamine can be synthesized from [1-¹³C]glucose (Figure 1B), but as just noted, this is apparently not a significant precursor of neuronal glutamate. In contrast, [4-¹³C]glutamate that is taken up by astrocytes, following release from neurons, can be used to synthesize [4-¹³C]glutamine (Figure 1B), and approximately 40% of [4-¹³C]glutamine is derived via this pathway (Hassel et al. 1997). Astrocytes can also convert [1,2-¹³C]acetate directly to [1,2-¹³C]acetyl CoA, and after several steps, produce [4,5-¹³C]glutamate and [4,5-¹³C]glutamine (Figure 1C). The [4,5-¹³C]glutamine is released and can be converted to [4,5-¹³C]glutamate in neurons. In GABAergic neurons this glutamate can be decarboxylated to [1,2-¹³C]GABA (Figure 1C). For simplicity only first turn isotopomers are shown in Figure 1. If α-[4-¹³C]ketoglutarate stays in the TCA cycle, [3-¹³C]/[2-¹³C]glutamate can be formed. For details on the isotopomers derived from the second turn see (Melo et al. 2006).

Figure 2

Increased GFAP expression in GFAP transgenic mice. Immunofluorescent staining shows dramatically elevated GFAP (red) in cortical subpial (A, B) and cerebellar (C, D) astrocytes in GFAP transgenic mice (B, D) compared with wild-type animals (A, C). DAPI stained nuclei are shown in blue. Images for wild-type and trangenic animals were taken with equivalent exposure and gain for the same regions. Scale bar = 50 µm.

Figure 3

Motor performance of GFAP transgenic mice (n=9) compared to littermate controls (n=8). A) Time spent on the rotarod increased across trials (indicating motor learning), but did not differ between transgenics and controls (Student’s t-test, p = 0.52). B) Transgenics exhibited significantly less grip strength of forepaws compared to controls (***,p < .001 by Student’s t-test). Error bars indicate ± 1 standard error of the mean.

Figure 4

Brain water in transgenic and control mice. Values in each experimental group represent percent brain water content of whole brain minus olfactory bulb. The results are expressed as mean ± SD and were analyzed with Student’s t-test. * p < 0.05.