Cellular and molecular outcomes of glutamine supplementation in the brain of succinic semialdehyde dehydrogenase-deficient mice

Abstract

Succinic semialdehyde dehydrogenase deficiency (SSADHD) manifests with low levels of glutamine in the brain, suggesting that central glutamine deficiency contributes to pathogenesis. Recently, we attempted to rescue the disease phenotype of aldh5a1 ^-/- mice, a murine model of SSADHD with dietary glutamine supplementation. No clinical rescue and no central glutamine improvement were observed. Here, we report the results of follow-up studies of the cellular and molecular basis of the resistance of the brain to glutamine supplementation. We first determined if the expression of genes involved in glutamine metabolism was impacted by glutamine feeding. We then searched for changes of brain histology in response to glutamine supplementation, with a focus on astrocytes, known regulators of glutamine synthesis in the brain. Glutamine supplementation significantly modified the expression of glutaminase (gls) (0.6-fold down), glutamine synthetase (glul) (1.5-fold up), and glutamine transporters (solute carrier family 7, member 5 [slc7a5], 2.5-fold up; slc38a2, 0.6-fold down). The number of GLUL-labeled cells was greater in the glutamine-supplemented group than in controls (P < .05). Reactive astrogliosis, a hallmark of brain inflammation in SSADHD, was confirmed. We observed a 2-fold stronger astrocyte staining in mutants than in wild-type controls (optical density/cell were 1.8 ± 0.08 in aldh5a1 ^-/- and 0.99 ± 0.06 in aldh5a1 ^+/+ ; P < .0001), and a 3-fold higher expression of gfap and vimentin. However, glutamine supplementation did not improve the histological and molecular signature of astrogliosis. Thus, glutamine supplementation impacts genes implicated in central glutamine homeostasis without improving reactive astrogliosis. The mechanisms underlying glutamine deficiency and its contribution to SSADHD pathogenesis remain unknown and should be the focus of future investigations.

Keywords: GABA; GHB; astrocyte; dietary supplementation; glutamine; knockout mice.

FIGURE 1

Glutamine metabolism and transport in brain. Arrows adjacent to metabolites indicate expression in SSADHD. Glutamine and GABA‐metabolites: GABA, γ‐aminobutyric acid; SSA, succinic semialdehyde; GHB, γ‐hydroxybutyrate; α‐KG, α‐ketoglutarate. Genes involved in GABA metabolism (shown in blue): glud1, glutamate dehydrogenase; gls, glutaminase; glul, glutamate‐ammonia ligase (glutamine synthetase); gad1, glutamate decarboxylase; abat, 4‐aminobutyrate aminotransferase (GABA transaminase); aldh5a1, aldehyde dehydrogenase family 5, subfamily A1 = SSADH (“X” indicates site of defect in SSADHD). Genes involved in glutamine transport (and respective protein transporter; shown in green): solute carrier family: slc1a5 = ASCT2; slc7a5 = LAT1; slc7a6 = γ + LAT2; slc38a1/2/3/5/7 = SNAT1/2/3/5/7. Reactive astrocyte/astrogliosis markers (red): gfap = glial fibrillary acidic protein; nes = nestin; vim = vimentin. BBB, blood‐brain barrier; SSADH, succinic semialdehyde dehydrogenase; SSADHD, succinic semialdehyde dehydrogenase deficiency; TCA cycle, tricarboxylic acid cycle = Krebs cycle

FIGURE 2

GABA‐ and glutamine‐related gene transcripts. A,B, Genotype differences (aldh5a1 ^+/+, wild‐type; aldh5a1 ^−/−, mutants). C,D, Diet differences (CD and GD). aldh5a1, aldehyde dehydrogenase family 5, subfamily A1; glud1, glutamate dehydrogenase; gls, glutaminase; glul, glutamate‐ammonia ligase or glutamine synthetase. Statistics analysis of variance (ANOVA) performed as described in Section 2. CD, control diet; GABA, γ‐aminobutyric acid; GD, glutamine diet

FIGURE 3

Glutamine transporter gene transcript. A‐D, Genotype differences (aldh5a1 ^+/+, wild‐type; aldh5a1 ^−/−, mutants). E,F, Diet differences (CD and GD). Slc1a5, solute carrier family 1, member 5; slc7a5, solute carrier family 7, member 5; slc38a2, solute carrier family 38, member 2. Statistics (ANOVA) performed as described in Section 2. CD, control diet; GD, glutamine diet

FIGURE 4

Morphology and density of hippocampal astrocytes in experimental groups. Representative GFAP‐stained sections illustrating astrogliosis in aldh5a1 ^−/− mice compared to aldh5a1 ^+/+ (CD = control diet, panels A and B; GD = glutamine diet; panels C and D). GFAP, glial fibrillary acidic protein

FIGURE 5

Brain cortical astrogliosis. A, Cortical GFAP labeling measurement zones: inner (closest to corpus callosum), mid 1, mid 2, mid 3, and outer zones (representative cortex section). B, Cortical GFAP labeling (optical density; mean + 1 SE) by zone, diet (CD and GD) and genotype (aldh5a1 ^+/+, aldh5a1 ^−/−). Note the significant astrocytic presence in the two zones closest to corpus callosum (inner and mid 1 zones) in aldh5a1 ^−/− mice. Statistics were performed as described in Section 2; *P < .05. CD, control diet; GD, glutamine diet; GFAP, glial fibrillary acidic protein

FIGURE 6

Relative gene expression for markers of astrogliosis. Transcripts significantly altered between genotype (aldh5a1 ^+/+ = wild‐type; aldh5a1 ^−/− = mutants): glial fibrillary acidic protein (gfap, A), and vimentin (vim, B). Statistics performed as described in Section 2