Journal: Life Metabolism

Article Title: IDH1 mutation inhibits differentiation of astrocytes and glioma cells with low oxoglutarate dehydrogenase expression by disturbing α-ketoglutarate-related metabolism and epigenetic modification

doi: 10.1093/lifemeta/loae002

Figure Lengend Snippet: OGDH expression is low in IDH-mutated gliomas. (a) Schematic representation of oxidative decarboxylation of α-KG by OGDH. mutIDH, mutant IDH; D-2HG, D-2-hydroxyglutarate; TCA cycle, tricarboxylic acid cycle. (b) mRNA levels of OGDH , FH , CS , and SDHB in ID H-WT and ID H-MU LGGs were analyzed according to TCGA data ( n = 92 in ID H-WT group, n = 416 in ID H-MU group). (c) OGDH, CS, SDHB, and FH protein levels in ID H-WT and ID H-MU glioma tissues, n = 6 for each group. (d) IHC staining (left) and statistical analysis (right) of OGDH in ID H-WT and ID H-MU glioma tissues. Scale bars are 20 μm, n = 84 in I D H-WT group, and n = 89 in ID H-MU group. (e) Western blot analysis showing OGDH protein expression in ID H1-WT and ID H1-MU U87 isogenic cells (U87-iso) (left), and U87-Teton and U251-Teton glioma cells with Dox-inducible overexpression of mutant IDH1 (right). Biologically independent experiments were repeated three times.

Article Snippet: IDH1-WT (ATCC Lot: 63710285) and IDH1 -mutant ( ID H1 R132 H ) (ATCC Lot: 70004232) U87 isogenic cells were acquired from American Type Culture Collection (ATCC).

Techniques: Expressing, Mutagenesis, Immunohistochemistry, Western Blot, Over Expression

Journal: Life Metabolism

Article Title: IDH1 mutation inhibits differentiation of astrocytes and glioma cells with low oxoglutarate dehydrogenase expression by disturbing α-ketoglutarate-related metabolism and epigenetic modification

doi: 10.1093/lifemeta/loae002

Figure Lengend Snippet: Astrocyte-specific mutant IDH1 knock-in impedes cortex development in mice. (a) Survival curves of Id h1-mu ( n = 20) and Id h1-wt ( n = 20) mice. Statistical significance was determined using a log-rank (Mantel–Cox) test. (b and c) The brain weights (b) and biparietal diameters (c) of Id h1-wt and Id h1-mu mice in NBs and at 2, 4, and 8 weeks of age. (d and e) Macroscopic views (d) and H&E staining (e) of brain sections in Id h1-wt and Id h1-mu mice at the indicated ages. Scale bars are 5 mm (d) and 1 mm (e), respectively. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA (b and c). * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: IDH1-WT (ATCC Lot: 63710285) and IDH1 -mutant ( ID H1 R132 H ) (ATCC Lot: 70004232) U87 isogenic cells were acquired from American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Knock-In, Staining

Journal: Life Metabolism

Article Title: IDH1 mutation inhibits differentiation of astrocytes and glioma cells with low oxoglutarate dehydrogenase expression by disturbing α-ketoglutarate-related metabolism and epigenetic modification

doi: 10.1093/lifemeta/loae002

Figure Lengend Snippet: The IDH1 mutation impedes the maturation of astrocytes. (a) The astrocytes in the cerebral cortex of Id h1-wt and Id h1-mu mice were detected by performing GFAP staining. Scale bars are 10 μm. Five independent experiments were performed. (b) The densities of mature astrocytes (with more than three branches) were calculated based on high-power fields (HPFs) at different ages, n = 5. (c) Average branch lengths of Id h1-wt and Id h1-mu astrocytes in the cortex section, n = 5. (d) Immunoblotting for GFAP, Nestin, and Sox2 in the brains of Id h1-wt and Id h1-mu mice at the indicated ages. Five independent experiments were performed. (e) Expression of GFAP, Nestin, and Sox2 in Id h1-wt and Id h1-mu astrocytes. (f) Growth curves of Id h1-wt and Id h1-mu astrocytes. (g) IF staining of GFAP in Id h1-wt and Id h1-mu astrocytes. Scale bars are 200 μm (top) and 50 μm (bottom), respectively. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA (b, c, f). * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: IDH1-WT (ATCC Lot: 63710285) and IDH1 -mutant ( ID H1 R132 H ) (ATCC Lot: 70004232) U87 isogenic cells were acquired from American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Staining, Western Blot, Expressing

Journal: Life Metabolism

Article Title: IDH1 mutation inhibits differentiation of astrocytes and glioma cells with low oxoglutarate dehydrogenase expression by disturbing α-ketoglutarate-related metabolism and epigenetic modification

doi: 10.1093/lifemeta/loae002

Figure Lengend Snippet: The IDH1 mutation disrupts α-KG homeostasis in astrocytes. (a) Changes in TCA cycle intermetabolites in Id h1-mu astrocytes (normalized to the levels in the Id h1-wt astrocytes), n = 6. (b) Expression of TCA cycle enzymes, including CS, OGDH, SDHB, and FH, in Id h1-mu and Id h1-wt astrocytes, n = 3. (c) Levels of representative amino acids in Id h1-mu astrocytes (normalized to the levels in Id h1-wt astrocytes), n = 6. (d and e) Gln consumption (d) amd ammonia production (e) in Id h1-mu and Id h1-wt astrocytes, n = 4. (f) Expression of GLUD1, GLS, GLUL, and BCAT1 in Id h1-mu and Id h1-wt astrocytes, n = 3. (g) BCAA consumption in Id h1-mu and Id h1-wt astrocytes, n = 4. (h–j) After the Id h1-mu and Id h1-wt astrocytes were treated with DM-α-KG or DMSO, the levels of GFAP, Nestin, and Sox2 (h), and H3K27me2, H3K27me3, and H3K4me3 (j) were detected through immunoblotting, and changes in 5-hmC were detected by IF staining (i). Scale bars are 20 μm. Biologically independent experiments were repeated three times in h–j. Data are represented as the mean ± SEM. Statistical significance was determined using an unpaired Student’s t -test (d and e), Mann–Whitney test (g), and one-way ANOVA (i). * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: IDH1-WT (ATCC Lot: 63710285) and IDH1 -mutant ( ID H1 R132 H ) (ATCC Lot: 70004232) U87 isogenic cells were acquired from American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Expressing, Western Blot, Staining, MANN-WHITNEY

Journal: Life Metabolism

Article Title: IDH1 mutation inhibits differentiation of astrocytes and glioma cells with low oxoglutarate dehydrogenase expression by disturbing α-ketoglutarate-related metabolism and epigenetic modification

doi: 10.1093/lifemeta/loae002

Figure Lengend Snippet: The IDH1 mutation blocks the differentiation of glioma cells with low OGDH expression by reducing α-KG levels. (a) Western blot showing GFAP, Nestin, CD133, and CD44 protein expression in ID H1-MU U87 isogenic cells (left), U87-Teton and U251-Teton glioma cells with Dox-inducible overexpression of mutant IDH1 (right). (b) Protein levels of GFAP, Nestin, CD133, and CD44 in ID H1-WT and ID H1-MU U87 cells after OGDH expression was knocked down. (c) Changes in TCA cycle intermetabolites in ID H1-MU U87 cells with silenced OGDH (normalized to the levels in the wild-type groups), n = 6. (d–f) Gln consumption (d), ammonia production (e), and BCAA consumption (f) in ID H1-WT and ID H1-MU U87 cells with silenced OGDH expression, n = 4. (g) Protein levels of GLUD1, GLS, GLUL, and BCAT1 in ID H1-WT and ID H1-MU U87 cells with silenced OGDH expression. (h and i) Representative images of IF staining for 5-hmC (h) and immunoblotting for H3K27me2, H3K27me3, and H3K4me3 (i) in ID H1-WT and ID H1-MU U87 cells with silenced OGDH expression. Scale bars, 20 μm. Biologically independent experiments were repeated three times in a, b, g, and i. Data are represented as the mean ± SEM. Statistical significance was determined using one-way ANOVA (c–f, h). * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: IDH1-WT (ATCC Lot: 63710285) and IDH1 -mutant ( ID H1 R132 H ) (ATCC Lot: 70004232) U87 isogenic cells were acquired from American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Expressing, Western Blot, Over Expression, Staining

Journal: Life Metabolism

Article Title: IDH1 mutation inhibits differentiation of astrocytes and glioma cells with low oxoglutarate dehydrogenase expression by disturbing α-ketoglutarate-related metabolism and epigenetic modification

doi: 10.1093/lifemeta/loae002

Figure Lengend Snippet: The IDH1 mutation leads to metabolic and epigenetic alterations in astrocytes and gliomas with low OGDH expression by disturbing α-KG homeostasis. OGDH is expressed at lower levels in astrocytes and IDH-mutated gliomas. Low OGDH expression promotes the maturation of astrocytes by increasing α-KG levels. Elevated α-KG can increase Gln production and BCAA transamination and promote the demethylation of DNA/histones and astrocyte maturation. Interestingly, knockdown of OGDH facilitates the differentiation of glioma cells, and this effect can be reversed by an IDH1 mutation. The IDH1 mutation further reduces α-KG levels, particularly in glioma cells with low OGDH expression, which coincides with increased glutaminolysis, decreased BCAA transamination, and DNA/histone hypermethylation. These findings suggest that low OGDH expression establishes a specific α-KG metabolic context, wherein the IDH1 mutation exerts a more significant impact on α-KG homeostasis, promoting glioma dedifferentiation.

Article Snippet: IDH1-WT (ATCC Lot: 63710285) and IDH1 -mutant ( ID H1 R132 H ) (ATCC Lot: 70004232) U87 isogenic cells were acquired from American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Expressing, Knockdown