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 1

An isogenic, patient-derived iPSC system to study L2HGDH deficiency.

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An isogenic, patient-derived iPSC system to study L2HGDH deficiency. (A)...
(A) L2HGDH genotypes of 2 L-2HGA patients and their parents. (B) Relative 2HG abundance in plasma from L-2HGA patients, parents, and unrelated subjects. (C) Volcano plot of plasma metabolites comparing patient 1 to unrelated subjects (n = 427; sibling excluded). A linear mixed-effects model was used to account for repeated measures per individual, with subject ID as a random effect. P values were adjusted for multiple testing (Benjamini-Hochberg), with statistical significance thresholds of adjusted P < 0.01 and fold change > 2 or < 0.5. (D) Volcano plot of fibroblast metabolites comparing patient 1 to 29 unrelated lines (sibling excluded), each profiled in quadruplicate. A mixed-effects model was used as in C, with cell line as the random effect. (E) L-2HG concentrations in neonatal control, adult control, and patient fibroblasts. (F) Alignment showing the pathogenic L2HGDH variant (c.829C>T, red) and edits (blue) introduced by the ssODN repair template. Electropherograms show unedited and biallelically corrected iPSC alleles. (G and H) L-2HG levels in PSCs (G) and NPCs (H) from control and patient lines with unedited or corrected L2HGDH alleles. In G, “hPSC” denotes human pluripotent stem cells and is used to collectively refer to the H9 human embryonic stem cell line and the patient-derived human iPSC lines. For B, E, G, and H, data are shown as box-and-whisker plots with jittered points (n = 4 biological replicates for E, G, and H); boxes represent the 25th–75th percentile, horizontal lines indicate medians, and whiskers extend to 1.5× the interquartile range. Significance was determined by 1-way ANOVA with Tukey’s HSD test.