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 2

L-2HG accumulation enhances NPC proliferation and self-renewal while inhibiting neuronal differentiation.

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L-2HG accumulation enhances NPC proliferation and self-renewal while inh...
(A) Growth curves of NPCs derived from H9 control, unedited, and corrected iPSCs. (B) Stereoscopic images of cortical spheroids at day 30 of differentiation. Scale bar: 1,000 μm. (C) Quantification of spheroid surface area at day 30. (D) Images of colonies formed from single NPCs. Scale bars: 2 mm (top), 100 μm (bottom). (E) Frequency of colony formation in single NPCs. (F) Immunostaining for MAP2 and β-III-tubulin in unedited, corrected, and corrected + 30 μM octyl-L-2HG (o-L-2HG) neurons at day 14. DAPI marks nuclei. Scale bar: 100 μm. (G) Quantification of neurite length from images. (H) GSEA plot showing enrichment of the REACTOME_NEURONAL_SYSTEM gene set in corrected versus unedited neurons. (I) Fura-2 ratio recordings of Ca2+ dynamics during 45 mM KCl application in neurons differentiated for 47 days (unedited: n = 29; corrected: n = 24). (J) Quantification of PAX6 signal intensity normalized to DAPI in unedited and corrected neurons. (K) UMAP of single-cell transcriptomes from day 45 cortical spheroids (unedited and corrected; 12,660 cells each). Annotated populations include progenitors (prog.), precursors (prec.), neurons (n.), astrocyte (astro.) lineage, radial (rad.) glia, intermediate (inter.) progenitors, excitatory (excit.) neurons, inhibitory (inhib.) neurons, interneurons, and oligodendrocyte progenitor cells (OPC). (L and M) Proportions of immature neurons (L) and excitatory neurons (M) among annotated populations. For A, C, E, G, J, L, and M, data are shown as mean ± SEM. Significance was determined by 2-way ANOVA for A, 1-way ANOVA with Tukey’s HSD test for C (n = 10 H9, n = 15 unedited, n = 10 corrected) and G (n = 96 unedited, n = 86 corrected, n = 108 corrected + o-L-2HG), and unpaired 2-tailed Student’s t test for E (n = 8), J (n = 679 unedited, n = 430 corrected), and L and M (n = 3 each).