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 6

c-MYC depletion restores neuronal differentiation in L2HGDH-deficient NPCs.

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c-MYC depletion restores neuronal differentiation in L2HGDH-deficient NP...
(A) Immunoblot of nuclear c-MYC and Neurogenin-2 (NGN2) in unedited patient 1 NPCs treated with DMSO or 20 μM nocodazole (Noc) for 7 days. TBP was used as a loading control. (B) Quantification of neurite lengths in patient 1 NPCs treated with DMSO or 20 μM Noc at the NPC stage and throughout 14 day neuronal differentiation. (C) Immunoblot of nuclear c-MYC and Neurogenin-2 in patient 1 NPCs transduced with lentivirus expressing control shRNA (shSCR) or shMYC. (D) Quantification of neurite lengths in patient 1 NPCs transduced with shSCR or shMYC. The dashed line denotes the mean neurite length in corrected neurons. (E) Representative recordings of intracellular Ca2+ dynamics in neurons transduced with shSCR (n = 29) or shMYC (n = 24), captured every 10 seconds using Fura-2 ratio imaging during 45 mM KCl application. Neurons were differentiated for 47 days. (F) Immunoblot of nuclear c-MYC and Neurogenin-2 in patient 1 NPCs treated with DMSO or 50 μM EN4 for the indicated durations. (G) Quantification of neurite lengths in patient 1 NPCs treated with DMSO, EN4 throughout differentiation, EN4 at the NPC stage and throughout differentiation, or EN4 only at the NPC stage. The dashed line indicates the corrected neuron mean. (H) Representative Ca2+ recordings in neurons treated with DMSO (n = 28) or 50 μM EN4 (n = 26), acquired as in E. For B, D, and G, neurite lengths were measured using the SNT plug-in in ImageJ. Data are shown as mean ± 1 SEM. Significance was determined by unpaired 2-tailed Student’s t test for B and D or 1-way ANOVA with Tukey’s HSD test for G.