Neuronal differentiation requires extensive metabolic remodeling to support increased energetic and biosynthetic demands. Here, we present an integrated multi-omics and functional characterization of metabolic transitions during early differentiation of human induced pluripotent stem cells (iPSCs) into excitatory cortical neurons using doxycycline-inducible overexpression of neurogenin-2 (NGN2). We analyzed parental iPSCs and induced neurons (iNs) at days 7 and 14 of differentiation, integrating gene expression profiling, label-free quantitative proteomics, high-resolution respirometry, fluorescence lifetime imaging microscopy (FLIM), and 13C₆-glucose metabolic flux analysis. Our data reveal progressive metabolic remodeling associated with neuronal maturation, including enhanced oxidative phosphorylation, increased mitochondrial content, and respiratory capacity. Proteomic analyses showed upregulation of mitochondrial and antioxidant pathways, while FLIM indicated a progressive increase in enzyme-bound NAD(P)H, consistent with a shift toward oxidative metabolism. Notably, 13C₆-glucose tracing revealed delayed labeling of the intracellular pool of fully labeled glucose and tricarboxylic acid cycle metabolites, together with enhanced labeling of pentose phosphate pathway intermediates and glutathione in iNs, indicating a shift toward biosynthetic and antioxidant glucose utilization during differentiation. Despite this enhancement in mitochondrial function, differentiated neurons maintained glycolytic activity, suggesting metabolic flexibility. Our results define the first week of differentiation as a critical window of metabolic specialization and establish NGN2-iPSC-derived cortical neurons as a versatile and well-characterized model system for investigating bioenergetic remodeling during early human neurodevelopment. It provides a robust foundation for mechanistic insights and high-throughput evaluation of metabolic pathways relevant to human disease.

Department of Pathophysiology 2nd Faculty of Medicine Charles University Prague Czech Republic

Department of Physiology Faculty of Science Charles University Prague Czech Republic

Laboratory of Bioenergetics Institute of Physiology of the Czech Academy of Sciences Prague Czech Republic

Laboratory of Biomathematics Institute of Physiology of the Czech Academy of Sciences Prague Czech Republic

Laboratory of Cell and Developmental Biology Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Laboratory of Developmental Epileptology Institute of Physiology of the Czech Academy of Sciences Prague Czech Republic

Laboratory of Metabolomics Institute of Physiology of the Czech Academy of Sciences Prague Czech Republic

Zobrazit více v PubMed

Schuchmann S, Buchheim K, Heinemann U, Hosten N, Buttgereit F (2005) Oxygen consumption and mitochondrial membrane potential indicate developmental adaptation in energy metabolism of rat cortical neurons. Eur J Neurosci 21(10):2721–2732. 10.1111/j.1460-9568.2005.04109.x PubMed DOI

Zheng X, Boyer L, Jin M, Mertens J, Kim Y, Ma L, Ma L, Hamm M et al (2016) Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation. Elife. 10.7554/eLife.13374 PubMed PMC

Agostini M, Romeo F, Inoue S, Niklison-Chirou MV, Elia AJ, Dinsdale D, Morone N, Knight RA et al (2016) Metabolic reprogramming during neuronal differentiation. Cell Death Differ 23(9):1502–1514. 10.1038/cdd.2016.36 PubMed PMC

Li D, Ding Z, Gui M, Hou Y, Xie K (2020) Metabolic enhancement of glycolysis and mitochondrial respiration are essential for neuronal differentiation. Cell Reprogram 22(6):291–299. 10.1089/cell.2020.0034 PubMed DOI

Goyal MS, Hawrylycz M, Miller JA, Snyder AZ, Raichle ME (2014) Aerobic glycolysis in the human brain is associated with development and neotenous gene expression. Cell Metab 19(1):49–57. 10.1016/j.cmet.2013.11.020 PubMed DOI PMC

Goyal MS, Raichle ME (2018) Glucose requirements of the developing human brain. J Pediatr Gastroenterol Nutr 66(Suppl 3):S46–S49. 10.1097/MPG.0000000000001875 PubMed DOI PMC

Steiner P (2019) Brain fuel utilization in the developing brain. Ann Nutr Metab 75(Suppl 1):8–18. 10.1159/000508054 PubMed DOI

Cunnane SC, Crawford MA (2014) Energetic and nutritional constraints on infant brain development: implications for brain expansion during human evolution. J Hum Evol 77:88–98. 10.1016/j.jhevol.2014.05.001 PubMed DOI

Kuzawa CW, Chugani HT, Grossman LI, Lipovich L, Muzik O, Hof PR, Wildman DE, Sherwood CC et al (2014) Metabolic costs and evolutionary implications of human brain development. Proc Natl Acad Sci U S A 111(36):13010–13015. 10.1073/pnas.1323099111 PubMed PMC

Ketschek A, Sainath R, Holland S, Gallo G (2021) The axonal glycolytic pathway contributes to sensory axon extension and growth cone dynamics. J Neurosci Off J Soc Neurosci 41(31):6637–6651. 10.1523/JNEUROSCI.0321-21.2021 PubMed DOI PMC

Galera-Monge T, Zurita-Díaz F, Canals I, Hansen MG, Rufián-Vázquez L, Ehinger JK, Elmér E, Martin MA et al (2020) Mitochondrial dysfunction and calcium dysregulation in Leigh syndrome induced pluripotent stem cell derived neurons. Int J Mol Sci. 10.3390/ijms21093191 PubMed PMC

Inak G, Rybak-Wolf A, Lisowski P, Pentimalli TM, Jüttner R, Glažar P, Uppal K, Bottani E et al (2021) Defective metabolic programming impairs early neuronal morphogenesis in neural cultures and an organoid model of Leigh syndrome. Nat Commun 12(1):1929. 10.1038/s41467-021-22117-z PubMed PMC

Geffroy G, Benyahia R, Frey S, Desquiret-Dumas V, Gueguen N, Bris C, Belal S, Inisan A et al (2018) The accumulation of assembly intermediates of the mitochondrial complex I matrix arm is reduced by limiting glucose uptake in a neuronal-like model of MELAS syndrome. Biochim Biophys Acta - Mol Basis Dis 1864(5):1596–1608. 10.1016/j.bbadis.2018.02.005 PubMed DOI

Bhattacharya S, Yin J, Huo W, Chaum E (2022) Modeling of mitochondrial bioenergetics and autophagy impairment in MELAS-mutant iPSC-derived retinal pigment epithelial cells. Stem Cell Res Ther 13(1):260. 10.1186/s13287-022-02937-6 PubMed DOI PMC

Kann O, Kovács R, Njunting M, Behrens CJ, Otáhal J, Lehmann T-N, Gabriel S, Heinemann U (2005) Metabolic dysfunction during neuronal activation in the ex vivo hippocampus from chronic epileptic rats and humans. Brain 128(10):2396–2407. 10.1093/brain/awh568 PubMed DOI

Otáhal J, Folbergrová J, Kovacs R, Kunz WS, Maggio N (2014) Epileptic focus and alteration of metabolism. 209–243. 10.1016/B978-0-12-418693-4.00009-1 PubMed

Kovács R, Gerevich Z, Friedman A, Otáhal J, Prager O, Gabriel S, Berndt N (2018) Bioenergetic mechanisms of seizure control. Front Cell Neurosci 12:335. 10.3389/fncel.2018.00335 PubMed DOI PMC

Zhang Y, Pak C, Han Y, Ahlenius H, Zhang Z, Chanda S, Marro S, Patzke C et al (2013) Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron 78(5):785–798. 10.1016/j.neuron.2013.05.029 PubMed PMC

Fernandopulle MS, Prestil R, Grunseich C, Wang C, Gan L, Ward ME (2018) Transcription factor–mediated differentiation of human iPSCs into neurons. Curr Protoc Cell Biol 79(1):e51. 10.1002/cpcb.51 PubMed DOI PMC

Lin H-C, He Z, Ebert S, Schörnig M, Santel M, Nikolova MT, Weigert A, Hevers W et al (2021) NGN2 induces diverse neuron types from human pluripotency. Stem Cell Reports 16(9):2118–2127. 10.1016/j.stemcr.2021.07.006 PubMed PMC

Shan X, Zhang A, Rezzonico MG, Tsai M-C, Sanchez-Priego C, Zhang Y, Chen MB, Choi M et al (2024) Fully defined NGN2 neuron protocol reveals diverse signatures of neuronal maturation. Cell Rep Methods 4(9):100858. 10.1016/j.crmeth.2024.100858 PubMed DOI PMC

Khayat-Khoei M, Ershadinia N, Oluigbo D, Sit T, Weiner H, Mierau S (2025) Human iPSC-derived neurogenin-2 (NGN2) cortical neurons develop functional connectivity and small-world network topology in vitro (P6–6.017). Neurology 104:7. 10.1212/WNL.0000000000212109

Varderidou-Minasian S, Verheijen BM, Schätzle P, Hoogenraad CC, Pasterkamp RJ, Altelaar M (2020) Deciphering the proteome dynamics during development of neurons derived from induced pluripotent stem cells. J Proteome Res 19(6):2391–2403. 10.1021/acs.jproteome.0c00070 PubMed DOI PMC

Zhou X, Lee Y-K, Li X, Kim H, Sanchez-Priego C, Han X, Tan H, Zhou S et al (2024) Integrated proteomics reveals autophagy landscape and an autophagy receptor controlling PKA-RI complex homeostasis in neurons. Nat Commun 15(1):3113. 10.1038/s41467-024-47440-z PubMed PMC

Lee GB, Mazli WNAB, Hao L (2024) Multiomics evaluation of human iPSCs and iPSC-derived neurons. J Proteome Res 23(8):3149–3160. 10.1021/acs.jproteome.3c00790 PubMed DOI PMC

Jensen AMG, Raska J, Fojtik P, Monti G, Lunding M, Bartova S, Pospisilova V, van der Lee SJ et al (2024) The SORL1 p. Y1816C variant causes impaired endosomal dimerization and autosomal dominant Alzheimer’s disease. Proc Natl Acad Sci U S A 121:37. 10.1073/pnas.2408262121 PubMed PMC

Chen G, Gulbranson DR, Hou Z, Bolin JM, Ruotti V, Probasco MD, Smuga-Otto K, Howden SE et al (2011) Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8(5):424–429. 10.1038/nmeth.1593 PubMed PMC

Bardy C, van den Hurk M, Eames T, Marchand C, Hernandez RV, Kellogg M, Gorris M, Galet B et al (2015) Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro. Proc Natl Acad Sci 112:20. 10.1073/pnas.1504393112 PubMed PMC

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. 10.1038/nmeth.2019 PubMed PMC

Hrckulak D, Janeckova L, Lanikova L, Kriz V, Horazna M, Babosova O, Vojtechova M, Galuskova K et al (2018) Wnt effector TCF4 is dispensable for Wnt signaling in human cancer cells. Genes 9(9):439. 10.3390/genes9090439 PubMed PMC

Čunátová K, Reguera DP, Vrbacký M, Fernández-Vizarra E, Ding S, Fearnley IM, Zeviani M, Houštěk J et al (2021) Loss of COX4I1 leads to combined respiratory chain deficiency and impaired mitochondrial protein synthesis. Cells. 10.3390/cells10020369 PubMed PMC

Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, Mann M, Cox J (2016) The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods 13(9):731–740. 10.1038/nmeth.3901 PubMed DOI

Mootha VK, Lindgren CM, Eriksson K-F, Subramanian A, Sihag S, Lehar J, Puigserver P, Carlsson E et al. (2003) PGC-1α-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 34(3):267–273. 10.1038/ng1180 PubMed

Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL et al (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci 102(43):15545–15550. 10.1073/pnas.0506580102 PubMed PMC

Liberzon A, Birger C, Thorvaldsdóttir H, Ghandi M, Mesirov JP, Tamayo P (2015) The molecular signatures database hallmark gene set collection. Cell Syst 1(6):417–425. 10.1016/j.cels.2015.12.004 PubMed DOI PMC

Kanehisa M, Furumichi M, Sato Y, Kawashima M, Ishiguro-Watanabe M (2023) KEGG for taxonomy-based analysis of pathways and genomes. Nucleic Acids Res 51(D1):D587–D592. 10.1093/nar/gkac963 PubMed DOI PMC

Carbon S, Douglass E, Good BM, Unni DR, Harris NL, Mungall CJ, Basu S, Chisholm RL et al (2021) The Gene Ontology resource: enriching a GOld mine. Nucleic Acids Res 49(D1):D325–D334. 10.1093/nar/gkaa1113 PubMed PMC

Müller F-J, Laurent LC, Kostka D, Ulitsky I, Williams R, Lu C, Park I-H, Rao MS et al (2008) Regulatory networks define phenotypic classes of human stem cell lines. Nature 455(7211):401–405. 10.1038/nature07213 PubMed PMC

Perez-Riverol Y, Bandla C, Kundu DJ, Kamatchinathan S, Bai J, Hewapathirana S, John NS, Prakash A et al (2025) The PRIDE database at 20 years: 2025 update. Nucleic Acids Res 53(D1):D543–D553. 10.1093/nar/gkae1011 PubMed PMC

Deutsch EW, Bandeira N, Perez-Riverol Y, Sharma V, Carver JJ, Mendoza L, Kundu DJ, Wang S et al (2023) The ProteomeXchange consortium at 10 years: 2023 update. Nucleic Acids Res 51(D1):D1539–D1548. 10.1093/nar/gkac1040 PubMed PMC

Čunátová K, Vrbacký M, Puertas-Frias G, Alán L, Vanišová M, Saucedo-Rodríguez MJ, Houštěk J, Fernández-Vizarra E et al (2024) Mitochondrial translation is the primary determinant of secondary mitochondrial complex I deficiencies. iScience 27(8):110560. 10.1016/j.isci.2024.110560 PubMed PMC

Esteras N, Blacker TS, Zherebtsov EA, Stelmashuk OA, Zhang Y, Wigley WC, Duchen MR, Dinkova-Kostova AT et al (2023) Nrf2 regulates glucose uptake and metabolism in neurons and astrocytes. Redox Biol 62:102672. 10.1016/j.redox.2023.102672 PubMed PMC

Connolly NMC, Theurey P, Adam-Vizi V, Bazan NG, Bernardi P, Bolaños JP, Culmsee C, Dawson VL et al (2018) Guidelines on experimental methods to assess mitochondrial dysfunction in cellular models of neurodegenerative diseases. Cell Death Differ 25(3):542–572. 10.1038/s41418-017-0020-4 PubMed PMC

Sun Y, Day RN, Periasamy A (2011) Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy. Nat Protoc 6(9):1324–1340. 10.1038/nprot.2011.364 PubMed DOI PMC

Cajka T, Hricko J, Rudl Kulhava L, Paucova M, Novakova M, Kuda O (2023) Optimization of mobile phase modifiers for fast LC-MS-based untargeted metabolomics and lipidomics. Int J Mol Sci 24(3):1987. 10.3390/ijms24031987 PubMed DOI PMC

Brejchova K, Rahm M, Benova A, Domanska V, Reyes-Gutierez P, Dzubanova M, Trubacova R, Vondrackova M et al (2025) Uncovering mechanisms of thiazolidinediones on osteogenesis and adipogenesis using spatial fluxomics. Metabolism 166:156157. 10.1016/j.metabol.2025.156157 PubMed

Lopez-Lengowski K, Kathuria A, Gerlovin K, Karmacharya R (2021) Co-culturing microglia and cortical neurons differentiated from human induced pluripotent stem cells. J Vis Exp 175. 10.3791/62480 PubMed PMC

Wang Q, Mou X, Cao H, Meng Q, Ma Y, Han P, Jiang J, Zhang H et al (2012) A novel xeno-free and feeder-cell-free system for human pluripotent stem cell culture. Protein Cell 3(1):51–59. 10.1007/s13238-012-2002-0 PubMed PMC

Zdrazilova L, Hansikova H, Gnaiger E (2022) Comparable respiratory activity in attached and suspended human fibroblasts. PLoS ONE 17(3):e0264496. 10.1371/journal.pone.0264496 PubMed DOI PMC

Gnaiger E (2003) Oxygen conformance of cellular respiration. 39–55. 10.1007/978-1-4419-8997-0_4 PubMed

Vernerova A, Garcia-Souza LF, Soucek O, Kostal M, Rehacek V, Kujovska Krcmova L, Gnaiger E, Sobotka O (2021) Mitochondrial respiration of platelets: comparison of isolation methods. Biomedicines. 10.3390/biomedicines9121859 PubMed DOI PMC

Bird DK, Yan L, Vrotsos KM, Eliceiri KW, Vaughan EM, Keely PJ, White JG, Ramanujam N (2005) Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH. Cancer Res 65(19):8766–8773. 10.1158/0008-5472.CAN-04-3922 PubMed DOI

Walsh AJ, Cook RS, Manning HC, Hicks DJ, Lafontant A, Arteaga CL, Skala MC (2013) Optical metabolic imaging identifies glycolytic levels, subtypes, and early-treatment response in breast cancer. Cancer Res 73(20):6164–6174. 10.1158/0008-5472.CAN-13-0527 PubMed DOI PMC

Song A, Zhao N, Hilpert DC, Perry C, Baur JA, Wallace DC, Schaefer PM (2024) Visualizing subcellular changes in the NAD(H) pool size versus redox state using fluorescence lifetime imaging microscopy of NADH. Commun Biol 7(1):428. 10.1038/s42003-024-06123-7 PubMed DOI PMC

Yang X, Ha G, Needleman DJ (2021) A coarse-grained NADH redox model enables inference of subcellular metabolic fluxes from fluorescence lifetime imaging. Elife. 10.7554/eLife.73808 PubMed DOI PMC

Stringari C, Nourse JL, Flanagan LA, Gratton E (2012) Phasor fluorescence lifetime microscopy of free and protein-bound NADH reveals neural stem cell differentiation potential. PLoS ONE 7(11):e48014. 10.1371/journal.pone.0048014 PubMed DOI PMC

Kolenc OI, Quinn KP (2019) Evaluating cell metabolism through autofluorescence imaging of NAD(P)H and FAD. Antioxid Redox Signal 30(6):875–889. 10.1089/ars.2017.7451 PubMed DOI PMC

Yoo HC, Yu YC, Sung Y, Han JM (2020) Glutamine reliance in cell metabolism. Exp Mol Med 52(9):1496–1516. 10.1038/s12276-020-00504-8 PubMed DOI PMC

Stoll EA, Makin R, Sweet IR, Trevelyan AJ, Miwa S, Horner PJ, Turnbull DM (2015) Neural stem cells in the adult subventricular zone oxidize fatty acids to produce energy and support neurogenic activity. Stem Cells 33(7):2306–2319. 10.1002/stem.2042 PubMed DOI PMC

Gao X, Zhao L, Liu S, Li Y, Xia S, Chen D, Wang M, Wu S et al (2019) γ-6-phosphogluconolactone, a byproduct of the oxidative pentose phosphate pathway, contributes to AMPK activation through inhibition of PP2A. Mol Cell 76(6):857-871.e9. 10.1016/j.molcel.2019.09.007 PubMed PMC

Uittenbogaard M, Brantner CA, Chiaramello A (2018) Epigenetic modifiers promote mitochondrial biogenesis and oxidative metabolism leading to enhanced differentiation of neuroprogenitor cells. Cell Death Dis 9(3):360. 10.1038/s41419-018-0396-1 PubMed DOI PMC

Gallo G (2024) Neuronal glycolysis: focus on developmental morphogenesis and localized subcellular functions. Commun Integr Biol 17(1):2343532. 10.1080/19420889.2024.2343532 PubMed DOI PMC

Stauch KL, Purnell PR, Fox HS (2014) Quantitative proteomics of synaptic and nonsynaptic mitochondria: insights for synaptic mitochondrial vulnerability. J Proteome Res 13(5):2620–2636. 10.1021/pr500295n PubMed DOI PMC

Masin L, Bergmans S, Van Dyck A, Farrow K, De Groef L, Moons L (2024) Local glycolysis supports injury-induced axonal regeneration. J Cell Biol. 10.1083/jcb.202402133 PubMed DOI PMC

Varum S, Rodrigues AS, Moura MB, Momcilovic O, Easley CA, Ramalho-Santos J, Van Houten B, Schatten G (2011) Energy metabolism in human pluripotent stem cells and their differentiated counterparts. PLoS ONE 6(6):e20914. 10.1371/journal.pone.0020914 PubMed DOI PMC

Sinenko SA, Tomilin AN (2023) Metabolic control of induced pluripotency. Front Cell Dev Biol 11:1328522. 10.3389/fcell.2023.1328522 PubMed DOI PMC

Lee J, Hong S-W, Kim M-J, Lim Y-M, Moon SJ, Kwon H, Park SE, Rhee E-J et al (2024) Inhibition of sodium-glucose cotransporter-2 during serum deprivation increases hepatic gluconeogenesis via the AMPK/AKT/FOXO signaling pathway. Endocrinol Metab 39(1):98–108. 10.3803/EnM.2023.1786 PubMed PMC

Shokati T, Zwingmann C, Leibfritz D (2005) Contribution of extracellular glutamine as an anaplerotic substrate to neuronal metabolism: a re-evaluation by multinuclear NMR spectroscopy in primary cultured neurons. Neurochem Res 30(10):1269–1281. 10.1007/s11064-005-8798-8 PubMed DOI

Yang C, Ko B, Hensley CT, Jiang L, Wasti AT, Kim J, Sudderth J, Calvaruso MA et al (2014) Glutamine oxidation maintains the TCA cycle and cell survival during impaired mitochondrial pyruvate transport. Mol Cell 56(3):414–424. 10.1016/j.molcel.2014.09.025 PubMed PMC

Knobloch M, Pilz G-A, Ghesquière B, Kovacs WJ, Wegleiter T, Moore DL, Hruzova M, Zamboni N et al (2017) A fatty acid oxidation-dependent metabolic shift regulates adult neural stem cell activity. Cell Rep 20(9):2144–2155. 10.1016/j.celrep.2017.08.029 PubMed PMC

Li H, Guglielmetti C, Sei YJ, Zilberter M, Le Page LM, Shields L, Yang J, Nguyen K et al (2023) Neurons require glucose uptake and glycolysis in vivo. Cell Rep 42(4):112335. 10.1016/j.celrep.2023.112335 PubMed PMC

Tajan M, Hennequart M, Cheung EC, Zani F, Hock AK, Legrave N, Maddocks ODK, Ridgway RA et al (2021) Serine synthesis pathway inhibition cooperates with dietary serine and glycine limitation for cancer therapy. Nat Commun 12(1):366. 10.1038/s41467-020-20223-y PubMed PMC

Aoyama K, Watabe M, Nakaki T (2008) Regulation of neuronal glutathione synthesis. J Pharmacol Sci 108(3):227–238. 10.1254/jphs.08r01cr PubMed DOI

Almeida AS, Soares NL, Sequeira CO, Pereira SA, Sonnewald U, Vieira HLA (2018) Improvement of neuronal differentiation by carbon monoxide: role of pentose phosphate pathway. Redox Biol 17:338–347. 10.1016/j.redox.2018.05.004 PubMed DOI PMC

Chen L, Zhang Z, Hoshino A, Zheng HD, Morley M, Arany Z, Rabinowitz JD (2019) NADPH production by the oxidative pentose-phosphate pathway supports folate metabolism. Nat Metab 1(3):404–415. 10.1038/s42255-019-0043-x PubMed DOI PMC

Iguchi Y, Katsuno M, Takagi S, Ishigaki S, Niwa J, Hasegawa M, Tanaka F, Sobue G (2012) Oxidative stress induced by glutathione depletion reproduces pathological modifications of TDP-43 linked to TDP-43 proteinopathies. Neurobiol Dis 45(3):862–870. 10.1016/j.nbd.2011.12.002 PubMed DOI

Won SJ, Kim J-E, Cittolin-Santos GF, Swanson RA (2015) Assessment at the single-cell level identifies neuronal glutathione depletion as both a cause and effect of ischemia-reperfusion oxidative stress. J Neurosci 35(18):7143–7152. 10.1523/JNEUROSCI.4826-14.2015 PubMed DOI PMC

Le Belle JE, Orozco NM, Paucar AA, Saxe JP, Mottahedeh J, Pyle AD, Wu H, Kornblum HI (2011) Proliferative neural stem cells have high endogenous ROS levels that regulate self-renewal and neurogenesis in a PI3K/Akt-dependant manner. Cell Stem Cell 8(1):59–71. 10.1016/j.stem.2010.11.028 PubMed DOI PMC

Shahin WS, Ebed SO, Tyler SR, Miljkovic B, Choi SH, Zhang Y, Zhou W, Evans IA et al (2023) Redox-dependent Igfbp2 signaling controls Brca1 DNA damage response to govern neural stem cell fate. Nat Commun 14(1):444. 10.1038/s41467-023-36174-z PubMed PMC

Schwartz EI, Smilenov LB, Price MA, Osredkar T, Baker RA, Ghosh S, Shi F-D, Vollmer TL et al (2007) Cell cycle activation in postmitotic neurons is essential for DNA repair. Cell Cycle 6(3):318–329. 10.4161/cc.6.3.3752 PubMed

Khacho M, Clark A, Svoboda DS, Azzi J, MacLaurin JG, Meghaizel C, Sesaki H, Lagace DC et al (2016) Mitochondrial dynamics impacts stem cell identity and fate decisions by regulating a nuclear transcriptional program. Cell Stem Cell 19(2):232–247. 10.1016/j.stem.2016.04.015 PubMed

He Y, Xie K, Yang K, Wang N, Zhang L (2025) Unraveling the interplay between metabolism and neurodevelopment in health and disease. CNS Neurosci Ther. 10.1111/cns.70427 PubMed DOI PMC

Spinazzola A, Zeviani M (2009) Disorders from perturbations of nuclear-mitochondrial intergenomic cross-talk. J Intern Med 265(2):174–192. 10.1111/j.1365-2796.2008.02059.x PubMed DOI

Alexeyev M, Shokolenko I, Wilson G, LeDoux S (2013) The maintenance of mitochondrial DNA integrity–critical analysis and update. Cold Spring Harb Perspect Biol 5(5):a012641–a012641. 10.1101/cshperspect.a012641 PubMed PMC

Prasad R, Çağlayan M, Dai D-P, Nadalutti CA, Zhao M-L, Gassman NR, Janoshazi AK, Stefanick DF et al (2017) DNA polymerase β: a missing link of the base excision repair machinery in mammalian mitochondria. DNA Repair 60:77–88. 10.1016/j.dnarep.2017.10.011 PubMed PMC

Garone C, De Giorgio F, Carli S (2024) Mitochondrial metabolism in neural stem cells and implications for neurodevelopmental and neurodegenerative diseases. J Transl Med 22(1):238. 10.1186/s12967-024-05041-w PubMed DOI PMC

Van Bergen NJ, Massey S, Stait T, Ellery M, Reljić B, Formosa LE, Quigley A, Dottori M et al (2021) Abnormalities of mitochondrial dynamics and bioenergetics in neuronal cells from CDKL5 deficiency disorder. Neurobiol Dis 155:105370. 10.1016/j.nbd.2021.105370 PubMed

Bogetofte H, Jensen P, Ryding M, Schmidt SI, Okarmus J, Ritter L, Worm CS, Hohnholt MC et al (2019) PARK2 mutation causes metabolic disturbances and impaired survival of human iPSC-derived neurons. Front Cell Neurosci. 10.3389/fncel.2019.00297 PubMed PMC

An MC, Zhang N, Scott G, Montoro D, Wittkop T, Mooney S, Melov S, Ellerby LM (2012) Genetic correction of Huntington’s disease phenotypes in induced pluripotent stem cells. Cell Stem Cell 11(2):253–263. 10.1016/j.stem.2012.04.026 PubMed DOI PMC

Huang C-W, Rust NC, Wu H-F, Yin A, Zeltner N, Yin H, Hart GW (2023) Low glucose induced Alzheimer’s disease-like biochemical changes in human induced pluripotent stem cell-derived neurons is due to dysregulated O-GlcNAcylation. Alzheimers Dement 19(11):4872–4885. 10.1002/alz.13058 PubMed DOI PMC

Müller Y, Lengacher L, Friscourt F, Quairiaux C, Stoppini L, Magistretti PJ, Lengacher S, Finsterwald C (2024) Epileptiform activity in brain organoids derived from patient with glucose transporter 1 deficiency syndrome. Front Neurosci 18:1498801. 10.3389/fnins.2024.1498801 PubMed DOI PMC

Thevenet J, De Marchi U, Domingo JS, Christinat N, Bultot L, Lefebvre G, Sakamoto K, Descombes P et al (2016) Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems. FASEB J 30(5):1913–1926. 10.1096/fj.201500182 PubMed

Shih P-Y, Kreir M, Kumar D, Seibt F, Pestana F, Schmid B, Holst B, Clausen C et al (2021) Development of a fully human assay combining NGN2-inducible neurons co-cultured with iPSC-derived astrocytes amenable for electrophysiological studies. Stem Cell Res 54:102386. 10.1016/j.scr.2021.102386 PubMed

Khadimallah I, Jenni R, Cabungcal J-H, Cleusix M, Fournier M, Beard E, Klauser P, Knebel J-F et al (2022) Mitochondrial, exosomal miR137-COX6A2 and gamma synchrony as biomarkers of parvalbumin interneurons, psychopathology, and neurocognition in schizophrenia. Mol Psychiatry 27(2):1192–1204. 10.1038/s41380-021-01313-9 PubMed PMC

Sasuclark AR, Watanabe M, Roshto K, Kilonzo VW, Zhang Y, Pitts MW (2025) Selenium deficiency impedes maturation of parvalbumin interneurons, perineuronal nets, and neural network activity. Redox Biol 81:103548. 10.1016/j.redox.2025.103548 PubMed DOI PMC

Gunhanlar N, Shpak G, van der Kroeg M, Gouty-Colomer LA, Munshi ST, Lendemeijer B, Ghazvini M, Dupont C et al (2018) A simplified protocol for differentiation of electrophysiologically mature neuronal networks from human induced pluripotent stem cells. Mol Psychiatry 23(5):1336–1344. 10.1038/mp.2017.56 PubMed PMC

Wattanapanitch M, Klincumhom N, Potirat P, Amornpisutt R, Lorthongpanich C, U-pratya Y, Laowtammathron C, Kheolamai P et al (2014) Dual small-molecule targeting of SMAD signaling stimulates human induced pluripotent stem cells toward neural lineages. PLoS ONE 9(9):e106952. 10.1371/journal.pone.0106952 PubMed PMC

Suzuki H, Egawa N, Imamura K, Kondo T, Enami T, Tsukita K, Suga M, Yada Y et al (2024) Mutant α-synuclein causes death of human cortical neurons via ERK1/2 and JNK activation. Mol Brain 17(1):14. 10.1186/s13041-024-01086-6 PubMed PMC

Mok RSF, Zhang W, Sheikh TI, Pradeepan K, Fernandes IR, DeJong LC, Benigno G, Hildebrandt MR et al (2022) Wide spectrum of neuronal and network phenotypes in human stem cell-derived excitatory neurons with Rett syndrome-associated MECP2 mutations. Transl Psychiatry 12(1):450. 10.1038/s41398-022-02216-1 PubMed PMC

Busskamp V, Lewis NE, Guye P, Ng AHM, Shipman SL, Byrne SM, Sanjana NE, Murn J et al (2014) Rapid neurogenesis through transcriptional activation in human stem cells. Mol Syst Biol 10(11):760. 10.15252/msb.20145508 PubMed PMC

Girardin S, Clément B, Ihle SJ, Weaver S, Petr JB, Mateus JC, Duru J, Krubner M et al (2022) Topologically controlled circuits of human iPSC-derived neurons for electrophysiology recordings. Lab Chip 22(7):1386–1403. 10.1039/D1LC01110C PubMed PMC

Bullmann T, Kaas T, Ritzau-Jost A, Wöhner A, Kirmann T, Rizalar FS, Holzer M, Nerlich J et al (2024) Human iPSC-derived neurons with reliable synapses and large presynaptic action potentials. J Neurosci 44(24):349–361. 10.1523/JNEUROSCI.0971-23.2024 PubMed PMC

Nehme R, Zuccaro E, Ghosh SD, Li C, Sherwood JL, Pietilainen O, Barrett LE, Limone F et al (2018) Combining NGN2 programming with developmental patterning generates human excitatory neurons with NMDAR-mediated synaptic transmission. Cell Rep 23(8):2509–2523. 10.1016/j.celrep.2018.04.066 PubMed PMC