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. 2011 Jan;31(1):90-101.
doi: 10.1038/jcbfm.2010.146. Epub 2010 Aug 25.

Brain glutamine synthesis requires neuronal-born aspartate as amino donor for glial glutamate formation

Beatriz Pardo  1 , Tiago B Rodrigues, Laura Contreras, Miguel Garzón, Irene Llorente-Folch, Keiko Kobayashi, Takeyori Saheki, Sebastian Cerdan, Jorgina Satrústegui
Affiliations

Brain glutamine synthesis requires neuronal-born aspartate as amino donor for glial glutamate formation

Beatriz Pardo et al. J Cereb Blood Flow Metab. 2011 Jan.

Abstract

The glutamate-glutamine cycle faces a drain of glutamate by oxidation, which is balanced by the anaplerotic synthesis of glutamate and glutamine in astrocytes. De novo synthesis of glutamate by astrocytes requires an amino group whose origin is unknown. The deficiency in Aralar/AGC1, the main mitochondrial carrier for aspartate-glutamate expressed in brain, results in a drastic fall in brain glutamine production but a modest decrease in brain glutamate levels, which is not due to decreases in neuronal or synaptosomal glutamate content. In vivo (13)C nuclear magnetic resonance labeling with (13)C(2)acetate or (1-(13)C) glucose showed that the drop in brain glutamine is due to a failure in glial glutamate synthesis. Aralar deficiency induces a decrease in aspartate content, an increase in lactate production, and lactate-to-pyruvate ratio in cultured neurons but not in cultured astrocytes, indicating that Aralar is only functional in neurons. We find that aspartate, but not other amino acids, increases glutamate synthesis in both control and aralar-deficient astrocytes, mainly by serving as amino donor. These findings suggest the existence of a neuron-to-astrocyte aspartate transcellular pathway required for astrocyte glutamate synthesis and subsequent glutamine formation. This pathway may provide a mechanism to transfer neuronal-born redox equivalents to mitochondria in astrocytes.

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Figures

Figure 1
Figure 1
Aralar immunolabeling in brain neurons and astrocytes. (A–C) Brain sections of Aralar+/+ mice (A–C) were probed against Aralar (green) (A) and Cox-I (red) (B) antibodies. Merged images are shown in (C), with nucleus marked with Topro-3 (blue). Scale bar, 10 μm. Electron microscopy analysis of Aralar immunolabeling in brain sections (D–F). (D) Prominent mitochondrial Aralar-immunogold (arrowheads) labeling in a neuronal somata, showing also some cytoplasmic gold particles. The neuron is adjacent to a glial cell (oligodendrocyte), showing the typical heterocromatic nucleus and electrodense cytoplasm, in which no Aralar immunolabeling is observed. Note the lack of labeling in the two large glial mitochondria (asterisk). Scale bar, 0.5 μm. (E) Arrowheads indicate Aralar-immunogold labeling on mitochondria within a dendrite. Where indicated (mit) two large unlabeled mitochondria appear in an astrocytic process (with an irregular contour marked with a dashed line), in the neuropil close to the Aralar-immunolabeled dendrite. Scale bar, 0.5 μm. (F) An astrocyte process showing several unlabeled mitochondria ensheathes a neuronal dendrite with one labeled mitochondria (arrowhead). The astrocyte shows a typical irregular sinuous contour and glial filaments (glial fil). Scale bar, 0.5 μm. Immunogold particles were counted in 220 electron micrographs at × 50,000 from 22 vibratome sections from five different mice. The number of particles counted in mitochondria or other regions was 731 or 210 in neurons, and 43 or 29 in glia, respectively.
Figure 2
Figure 2
Lack of 13C-labeled glutamine in Aralar−/− mice. (A) Expansions of representative proton-decoupled 13C nuclear magnetic resonance spectra (125.13 MHz, 25°C, pH 7.2) of neutralized perchloric acid extracts from the cerebral tissue of control (Aralar+/+) and Aralar-deficient (Aralar−/−) mice intraperitoneally injected with (1,2-13C2) acetate. Only the 26 to 56 p.p.m. region is shown, with insets highlighting the 30 to 36 p.p.m. region. Asp, aspartate (C2: 53.0 p.p.m.; C3: 37.8 p.p.m.); GABA, γ-aminobutyric acid (C2: 35.4 p.p.m., C4: 40.4 p.p.m.); Gln, glutamine (C2: 55.0 p.p.m.; C3: 27.0 p.p.m.; C4: 31.6 p.p.m.); Glu, glutamate (C2: 55.4 p.p.m.; C3: 27.7 p.p.m.; C4: 34.2 p.p.m.); NAA, N-acetylaspartic acid (C2: 54.2 p.p.m.); PCr, phosphocreatine (C4′: 54.4 p.p.m.); p.p.m., parts per million; Tau, taurine (C1: 48.4 p.p.m.; C2: 36.1 p.p.m.). (B) Combined fractional 13C enrichment of glutamate C4 and glutamine C4, of extracts from the cerebral tissue of control (Aralar+/+) and Aralar-deficient (Aralar−/−) mice injected with (1,2-13C2) acetate or with (1-13C) glucose, determined as indicated in the text. See Cerdán et al (1990) for assignments of singlets and doublets. Arrows indicate the absence of Gln labeling in the KO mice extracts. Results are expressed as the mean±s.e.m.
Figure 3
Figure 3
Aspartate promotes glutamate synthesis in astroglial cell cultures. (A, B) Cortical astrocytes cultures from wild-type (WT) mice (DIV14) were incubated for 1 hour in KRBH (140 mmol/L NaCl, 3.6 mmol/L KCl, 0.5 mmol/L NaH2PO4, 0.5 mmol/L MgSO4, 1.5 mmol/L CaCl2, 2 mmol/L NaHCO3, 10 mmol/L Hepes, pH 7.4) containing 2 mmol/L glucose in the absence or presence of supplemented amino acids (γ-aminobutyric acid (GABA), aspartate, alanine, or leucine; 10 to 200 μmol/L). Cellular extracts and media were separately recovered to measure glutamate (A) and glutamine (B) content, respectively, by an enzymatic end point method. (C, D) Cortical astrocytes from WT mice (DIV14) were incubated for 1 hour in KRBH-2 mmol/L glucose (Ctr) and in the presence of added aspartate (Asp; 50 μmol/L), glutamate (Glu; 50 μmol/L) or both together (Asp/Glu). Cellular extracts and media were separately recovered to measure glutamate (C) and glutamine (D) content, respectively, as described above. Under those conditions, astroglial cultures were viable as detected by using the calcein-acetoxy methylester/propidium iodide essay. (E, F) Cortical astrocytes from WT and Aralar-KO mice (DIV14) were incubated for 2 hours in KRBH containing 15 mmol/L glucose in the absence or presence of 100 μmol/L aspartate, 100 μmol/L alanine, or 100 μmol/L leucine. Cellular extracts and media were separately recovered to measure glutamate (E) and glutamine (F) content, respectively, as described. Intracellular glutamate (A, C, E) and extracellular glutamine content (B, D, F) are expressed as nmol/mg protein. Results are mean±s.e.m. (n=6) of three independent experiments. Data were statistically evaluated by one-way analysis of variance followed by Student–Newman–Keuls's t-test method (***P⩽0.001; **P⩽0.01; *P⩽0.05). KRBH, Krebs-Ringer bicarbonate-HEPES buffer.
Figure 4
Figure 4
Neuron-to-glia transcelullar aspartate efflux pathway for glial glutamate synthesis. Neuronal mitochondria are provided with Aralar/AGC1/Slc25a12 and the oxoglutarate carrier/OGC/Slc25a11 and carry out the malate-aspartate shuttle to transfer NADH reducing equivalents to the mitochondrial matrix. AGC1 is irreversible in polarized mitochondria and the main pathway of glutamate supply to the mitochondrial matrix. As cAST functions in the direction of glutamate formation in cells with an active malate-aspartate shuttle, mitochondria are the only site where aspartate is produced (in the mitochondrial aspartate aminotransferase reaction), and aspartate leaves the matrix through AGC1 to reach the cytosol. De novo glutamate synthesis in astroglial cells takes place in the cytosol in the cAST reaction with aspartate as amino-nitrogen donor to α-KG. A second amino group (possibly arising from ammonia itself formed in neurons in the phosphate-activated glutaminase reaction) is acquired in the glutamine synthetase reaction and glial glutamine is now transferred to neurons along the glutamate–glutamine cycle (not shown). Oxaloacetate (OAA) arising from the cAST reaction is converted to malate, and malate entry in glial mitochondria along the OGC provides an alternative pathway for redox transfer to mitochondria, which partly compensates for the lack of a malate-aspartate shuttle in brain astrocytes. In this way, equivalent transfer to astroglial mitochondria is stoichiometrically related to de novo glutamate production. Alternatively, malate formed in astroglial cytosol may be transferred back to neurons, as malate is released to a higher extent from cultured astrocytes than from cultured neurons (Westergaard et al, 1994) (not shown). The presence of mitochondria in the fine peridentritic processes of astrocytes (Figures 1E and 1F) indicates astrocytic oxidative capability near synapses. This confirms the reports by the Nedergaard group (Lovatt et al, 2007) and suggests that astrocytes need not be predominately glycolytic to supply their energy during brain activation. Indeed, the labeling of Asp C3 (Supplementary Figure 4) by [1-13C]glucose and its dilution by unlabeled AcCoA in the [1,2-13C]acetate study indicates that astrocytes oxidize glucose and must have some redox carrier system. AGC, aspartate–glutamate carrier; Asp, aspartate; Gln, glutamine; Glu, glutamate; α-KG, α-ketoglutarate; Mal, malate; OAA, oxalacetic acid; OGC, α-ketoglutarate–malate carrier; Pyr, pyruvate.
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