l-2-Hydroxyglutarate impairs neuronal differentiation through epigenetic activation of MYC expression
Wen Gu, Xun Wang, Ashley Solmonson, Ling Cai, Yi Xiao, Alpaslan Tasdogan, Jordan Franklin, Yuannyu Zhang, Hua Zhang, Aundrea K. Westfall, Ashley Rowe, Hetali Trivedi, Brandon Faubert, Zheng Wu, Jessica Sudderth, Lauren G. Zacharias, Bushra Afroze, Ilya Bezprozvanny, Sunil Sudarshan, Feng Cai, Samuel K. McBrayer, Thomas P. Mathews, Ralph J. DeBerardinis
Wen Gu, Xun Wang, Ashley Solmonson, Ling Cai, Yi Xiao, Alpaslan Tasdogan, Jordan Franklin, Yuannyu Zhang, Hua Zhang, Aundrea K. Westfall, Ashley Rowe, Hetali Trivedi, Brandon Faubert, Zheng Wu, Jessica Sudderth, Lauren G. Zacharias, Bushra Afroze, Ilya Bezprozvanny, Sunil Sudarshan, Feng Cai, Samuel K. McBrayer, Thomas P. Mathews, Ralph J. DeBerardinis
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Research Article Clinical Research Development Metabolism

l-2-Hydroxyglutarate impairs neuronal differentiation through epigenetic activation of MYC expression

Abstract

High levels of l- and d-2-hydroxyglutarate (2HG), the reduced forms of α-ketoglutarate (αKG), are implicated in neurodevelopmental disorders and cancer by modulating αKG-dependent dioxygenases involved in histone, DNA, and RNA demethylation. L-2HG dehydrogenase (L2HGDH) deficiency, a rare autosomal recessive inborn error of metabolism associated with systemic L-2HG elevation, causes progressive neurological disability and increased brain tumor risk of unclear mechanism. Using an isogenic, patient-derived induced pluripotent stem cell system, we examined the impact of L2HGDH deficiency on neural progenitor cell (NPC) function and neuronal differentiation. L2HGDH deficiency caused L-2HG accumulation, NPC hyperproliferation, increased clonogenicity, and defective neuronal differentiation in 2D cultures and cortical spheroids. Editing the L2HGDH locus to WT reversed these effects. Inhibiting glutaminase reduced L-2HG levels and induced neuronal differentiation. L-2HG–dependent inhibition of KDM5 histone demethylases led to widespread retention of H3K4me2/3, markers of active gene expression, with prominent enrichment at the MYC locus and elevated MYC expression across multiple neural cell types. Despite broadly altered histone methylation, genetically or pharmacologically normalizing MYC completely restored neuronal differentiation. These data indicated that a primary metabolic disturbance activated MYC to favor self-renewal and suppress neuronal lineage commitment.

Authors

Wen Gu, Xun Wang, Ashley Solmonson, Ling Cai, Yi Xiao, Alpaslan Tasdogan, Jordan Franklin, Yuannyu Zhang, Hua Zhang, Aundrea K. Westfall, Ashley Rowe, Hetali Trivedi, Brandon Faubert, Zheng Wu, Jessica Sudderth, Lauren G. Zacharias, Bushra Afroze, Ilya Bezprozvanny, Sunil Sudarshan, Feng Cai, Samuel K. McBrayer, Thomas P. Mathews, Ralph J. DeBerardinis

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Figure 3

Suppressing L-2HG synthesis improves neurite formation in L2HGDH-deficient NPCs.

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Suppressing L-2HG synthesis improves neurite formation in L2HGDH-deficie...
(A) Schematic of 13C-labeling routes to L-2HG from [U-13C]glucose (blue) or [U-13C]glutamine (green) in NPCs. Glc, glucose; Pyr, pyruvate; Ac-CoA, acetyl-CoA; OAA, oxaloacetate; Cit, citrate; Gln, glutamine; GLS, glutaminase; GLSi, glutaminase inhibitor (CB-839); Glu, glutamate. (B) Ion counts of 13C-labeled 2HG isotopologues in NPCs derived from H9, control hiPSC, and patients 1 and 2 after 4 hours of labeling with [U-13C]glucose or [U-13C]glutamine. (C) Ion counts of 13C-labeled 2HG isotopologues in control hiPSC-derived and patient 1 NPCs cultured in [U-13C]glutamine and treated with DMSO or 1 μM CB-839 (GLSi) for 4 hours. (D) Immunofluorescence for MAP2 and β-III-tubulin in patient 1 NPCs after 14 days of neuronal differentiation, treated with DMSO, 30 μM octyl-L-2HG (o-L-2HG), 1 μM CB-839 (GLSi), or both. DAPI marks nuclei. Scale bar: 100 μm. (E) Quantification of total neurite length from cells in D, measured with the SNT plug-in in ImageJ. For B and C, ion counts were normalized to total ion current. Data were non-normally distributed (Shapiro-Wilk test), so statistical significance was assessed by Kruskal-Wallis test with Dunn’s post hoc correction (n = 4 per group). Significance was determined by 1-way ANOVA with Tukey’s HSD test for E (n = 90 for DMSO + DMSO, n = 116 for DMSO + o-L-2HG, n = 89 for GLSi + DMSO, n = 101 for GLSi + o-L-2HG). Data are shown as mean ± 1 SEM.