Mitochondrial respiration of a primary mixed culture of neurons from hippocampus at various stages of differentiation

Abstract

BACKGROUND: Primary mixed culture of neurons is often used to evaluate the molecular mechanisms underlying neurodegenerative disorders. However, isolating cells from the brain is accompanied by profound changes in cell morphology, behavior, and metabolic rate due to enzymatic disintegration and microenvironmental changes in cells. In this regard, the mitochondrial basal respiration of neurons should be considered as an indicator of the formation of functional activity of the cell.

AIM: To evaluate mitochondrial respiration in a primary mixed culture of hippocampal neurons at various stages of differentiation.

METHODS: This study included mice of line CD-1 and was conducted in two series: the first studied the primary mixed embryonic culture on the 18^th day of gestation (E18) and the second used the postnatal culture on the 2^nd day after birth (P2). A Seahorse XF HS Mini (Agilent, USA) was used to measure the functional parameters of cell metabolism. The oxygen consumption rate was calculated based on the results of the mitochondrial respiration profile of the primary mixed culture built during its differentiation.

RESULTS: On the 5^th day of neuronal differentiation in the culture, maximum mitochondrial respiration was established both in the hippocampal culture obtained from embryos on the 18^th day of gestation and in the culture taken from mice on the 2^nd day after birth. As cells differentiated in culture from days 2 to 11, the substrate oxidation rate increased almost twofold in the culture of hippocampal neurons obtained from embryos, which increased the metabolic potential.

CONCLUSION: The study results prove that the hippocampus can be used to study the role of mitochondria in neurogenesis.

Keywords

hippocampus / neurons / embryo culture / postnatal culture / cell respiration

References

[1] Rangaraju V, Calloway N, Ryan TA. Activity-driven local ATP synthesis is required for synaptic function. Cell. 2014;156(4):825–835. doi: 10.1016/j.cell.2013.12.042

[2] Rangaraju V., Calloway N., Ryan T.A. Activity-driven local ATP synthesis is required for synaptic function // Cell. 2014. Vol. 156, N 4. P. 825–835. doi: 10.1016/j.cell.2013.12.042

[3] Pathak D, Shields LY, Mendelsohn BA, et al. The role of mitochondrially derived ATP in synaptic vesicle recycling. J Biol Chem. 2015;290(37):22325–22336. doi: 10.1074/jbc.M115.656405

[4] Pathak D., Shields L.Y., Mendelsohn B.A., et al. The role of mitochondrially derived ATP in synaptic vesicle recycling // J Biol Chem. 2015. Vol. 290, N 37. P. 22325–22336. doi: 10.1074/jbc.M115.656405

[5] Princz A, Kounakis K, Tavernarakis N. Mitochondrial contributions to neuronal development and function. Biol Chem. 2018;399(7):723–739. doi: 10.1515/hsz-2017-0333

[6] Princz A., Kounakis K., Tavernarakis N. Mitochondrial contributions to neuronal development and function // Biol Chem. 2018. Vol. 399, N 7. P. 723–739. doi: 10.1515/hsz-2017-0333

[7] Saha PP, Vishwanathan V, Bankapalli K, D’Silva P. Iron-sulfur protein assembly in human cells. Rev Physiol Biochem Pharmacol. 2018;174:25–65. doi: 10.1007/112_2017_5

[8] Saha P.P., Vishwanathan V., Bankapalli K., D’Silva P. Iron-sulfur protein assembly in human cells // Rev Physiol Biochem Pharmacol. 2018. Vol. 174. P. 25–65. doi: 10.1007/112_2017_5

[9] Ghosh N, Sil PC. Mitochondria and apoptosis. In: de Oliveira MR, editors. Mitochondrial physiology and vegetal molecules. Academic Press; 2021. P. 127–149.

[10] Ghosh N., Sil P.C. Mitochondria and apoptosis. In: de Oliveira M.R., editors. Mitochondrial physiology and vegetal molecules. Academic Press, 2021. P. 127-149.

[11] Pinton P, Leo S, Wieckowski MR, et al. Long-term modulation of mitochondrial Ca2+ signals by protein kinase C isozymes. J Cell Biol. 2004;165(2):223–232. doi: 10.1083/jcb.200311061

[12] Pinton P., Leo S, Wieckowski M.R., et al. Long-term modulation of mitochondrial Ca2+ signals by protein kinase C isozymes // J Cell Biol. 2004. Vol. 165, N 2. P. 223–232. doi: 10.1083/jcb.200311061

[13] Giorgi C, Missiroli S, Patergnani S, et al. Mitochondria-associated membranes: composition, molecular mechanisms, and physiopathological implications. Antioxid Redox Signal. 2015;22(12):995–1019. doi: 10.1089/ars.2014.6223

[14] Giorgi C., Missiroli S., Patergnani S., et al. Mitochondria-associated membranes: composition, molecular mechanisms, and physiopathological implications // Antioxid Redox Signal. 2015. Vol. 22, N 12. P. 995–1019. doi: 10.1089/ars.2014.6223

[15] West AP, Shadel GS. Mitochondrial DNA in innate immune responses and inflammatory pathology. Nat Rev Immunol. 2017;17(6):363–375. doi: 10.1038/nri.2017.21

[16] West A.P., Shadel G.S. Mitochondrial DNA in innate immune responses and inflammatory pathology // Nat Rev Immunol. 2017. Vol. 17, N. 6. P. 363–375. doi: 10.1038/nri.2017.21

[17] Slanzi A, Iannoto G, Rossi B, et al. In vitro models of neurodegenerative diseases. Front Cell Dev Biol. 2020;8:328. doi: 10.3389/fcell.2020.00328

[18] Slanzi A., Iannoto G., Rossi B., et al. In vitro models of neurodegenerative diseases // Front Cell Dev Biol. 2020. Vol. 8. P. 328. doi: 10.3389/fcell.2020.00328

[19] 1Faria-Pereira A, Temido-Ferreira M, Morais VA. brainphys neuronal media support physiological function of mitochondria in mouse primary neuronal cultures. Front Mol Neurosci. 2022;15:837448. doi: 10.3389/fnmol.2022.837448

[20] Faria-Pereira A., Temido-Ferreira M., Morais V.A. BrainPhys neuronal media support physiological function of mitochondria in mouse primary neuronal cultures // Front Mol Neurosci. 2022. Vol. 15. P. 837448. doi: 10.3389/fnmol.2022.837448

[21] Xu X, Duan S, Yi F, et al. Mitochondrial regulation in pluripotent stem cells. Cell Metab. 2013;18(3):325–332. doi: 10.1016/j.cmet.2013.06.005

[22] Xu X., Duan S., Yi F., et al. Mitochondrial regulation in pluripotent stem cells // Cell Metab. 2013. Vol. 18, N 3. P. 325–332. doi: 10.1016/j.cmet.2013.06.005

[23] Chen CT, Hsu SH, Wei YH. Upregulation of mitochondrial function and antioxidant defense in the differentiation of stem cells. Biochim Biophys Acta. 2010;1800(3):257–263. doi: 10.1016/j.bbagen.2009.09.001

[24] Chen C.T., Hsu S.H., Wei Y.H. Upregulation of mitochondrial function and antioxidant defense in the differentiation of stem cells // Biochim Biophys Acta. 2010. Vol. 1800, N 3. P. 257–263. doi: 10.1016/j.bbagen.2009.09.001

[25] Folmes CDL, Nelson TJ, Dzeja PP, Terzic A. Energy metabolism plasticity enables stemness programs. Ann N Y Acad Sci. 2012;1254:82–89. doi: 10.1111/j.1749-6632.2012.06487.x

[26] Folmes C.D.L., Nelson T.J., Dzeja P.P., Terzic A. Energy metabolism plasticity enables stemness programs // Ann N Y Acad Sci. 2012. Vol. 1254. P. 82–89. doi: 10.1111/j.1749-6632.2012.06487.x

[27] Patent RUS N 2802254 С1. Zelentsova AS, Borisova AIu, Shmigerova VS, et al. Method of cultivating a primary mixed culture of neurons for assessing mitochondrial respiration. EDN: VJREHE

[28] Патент РФ на изобретение № 2802254 С1. Способ культивирования первичной смешанной культуры нейронов для оценки митохондриального дыхания. Зеленцова А.С., Борисова А.Ю., Шмигерова В.С., и др. EDN: VJREHE

[29] Alvarez-Buylla A, Seri B, Doetsch F. Identification of neural stem cells in the adult vertebrate brain. Brain Res Bull. 2002;57(6):751–758. doi: 10.1016/s0361-9230(01)00770-5

[30] Alvarez-Buylla A., Seri B., Doetsch F. Identification of neural stem cells in the adult vertebrate brain // Brain Res Bull. 2002. Vol. 57, N 6. P. 751–758. doi: 10.1016/s0361-9230(01)00770-5

[31] Busche MA. In vivo two-photon calcium imaging of hippocampal neurons in Alzheimer mouse models. Methods Mol Biol. 2018;1750:341–351. doi: 10.1007/978-1-4939-7704-8_23

[32] Busche M.A. In vivo two-photon calcium imaging of hippocampal neurons in Alzheimer mouse models // Methods Mol Biol. 2018. Vol. 1750. P. 341–351. doi: 10.1007/978-1-4939-7704-8_23

[33] Koyama R, Ikegaya Y. The molecular and cellular mechanisms of axon guidance in mossy fiber sprouting. Front Neurol. 2018;9:382. doi: 10.3389/fneur.2018.00382

[34] Koyama R., Ikegaya Y. The molecular and cellular mechanisms of axon guidance in mossy fiber sprouting // Front Neurol. 2018. Vol. 9. P. 382. doi: 10.3389/fneur.2018.00382

[35] Wu Q, Takano H, Riddle DM, et al. α-synuclein (αSyn) preformed fibrils induce endogenous αSyn aggregation, compromise synaptic activity and enhance synapse loss in cultured excitatory hippocampal neurons. J Neurosci. 2019;39(26):5080–5094. doi: 10.1523/JNEUROSCI.0060-19.2019

[36] Wu Q., Takano H., Riddle D.M., et al. α-synuclein (αSyn) preformed fibrils induce endogenous αSyn aggregation, compromise synaptic activity and enhance synapse loss in cultured excitatory hippocampal neurons // J Neurosci. 2019. Vol. 39, N 26. P. 5080–5094. doi: 10.1523/JNEUROSCI.0060-19.2019

[37] Rush T, Roth JR, Thompson SJ, et al. A peptide inhibitor of Tau-SH3 interactions ameliorates amyloid-beta toxicity. Neurobiol Dis. 2020;134:104668. doi: 10.1016/j.nbd.2019.104668

[38] Rush T., Roth J.R., Thompson S.J., et al. A peptide inhibitor of Tau-SH3 interactions ameliorates amyloid-beta toxicity // Neurobiol Dis. 2020. Vol. 134. P. 104668. doi: 10.1016/j.nbd.2019.104668

[39] Kaech S, Banker G. Culturing hippocampal neurons. Nat Protoc. 2006;1(5):2406–2415. doi: 10.1038/nprot.2006.356

[40] Kaech S., Banker G. Culturing hippocampal neurons // Nat Protoc. 2006. Vol. 1, N 5. P. 2406–2415. doi: 10.1038/nprot.2006.356

[41] Brewer GJ, Torricelli JR. Isolation and culture of adult neurons and neurospheres. Nat Protoc. 2007;2(6):1490–1498. doi: 10.1038/nprot.2007.207

[42] Brewer G.J., Torricelli J.R. Isolation and culture of adult neurons and neurospheres // Nat Protoc. 2007. Vol. 2, N. 6. P. 1490–1498. doi: 10.1038/nprot.2007.207

[43] Zhang H, Xing L, Rossoll W, et al. Multiprotein complexes of the survival of motor neuron protein SMN with Gemins traffic to neuronal processes and growth cones of motor neurons. J Neurosci. 2006;26(33):8622–8632. doi: 10.1523/JNEUROSCI.3967-05.2006

[44] Zhang H., Xing L., Rossoll W., et al. Multiprotein complexes of the survival of motor neuron protein SMN with Gemins traffic to neuronal processes and growth cones of motor neurons // J Neurosci. 2006. Vol. 26, N 33. P. 8622–8632. doi: 10.1523/JNEUROSCI.3967-05.2006

[45] Brewer KL, Yezierski RP. Effects of adrenal medullary transplants on pain-related behaviors following excitotoxic spinal cord injury. Brain Res. 1998;798(1-2):83–92. doi: 10.1016/s0006-8993(98)00398-9

[46] Brewer K.L., Yezierski R.P. Effects of adrenal medullary transplants on pain-related behaviors following excitotoxic spinal cord injury // Brain Res. 1998. Vol. 798, N 1-2. P. 83–92. doi: 10.1016/s0006-8993(98)00398-9

[47] Brewer LD, Thibault O, Staton J, et al. Increased vulnerability of hippocampal neurons with age in culture: temporal association with increases in NMDA receptor current, NR2A subunit expression and recruitment of L-type calcium channels. Brain Res. 2007;1151:20–31. doi: 10.1016/j.brainres.2007.03.020

[48] Brewer L.D., Thibault O., Staton J., et al. Increased vulnerability of hippocampal neurons with age in culture: temporal association with increases in NMDA receptor current, NR2A subunit expression and recruitment of L-type calcium channels // Brain Res. 2007. Vol. 1151. P. 20–31. doi: 10.1016/j.brainres.2007.03.020

[49] Moutin E, Hemonnot AL, Seube V, et al. Procedures for culturing and genetically manipulating murine hippocampal postnatal neurons. Front Synaptic Neurosci. 2020;12:19. doi: 10.3389/fnsyn.2020.00019

[50] Moutin E., Hemonnot A.L., Seube V., et al. Procedures for culturing and genetically manipulating murine hippocampal postnatal neurons // Front Synaptic Neurosci. 2020. Vol. 12. P. 19. doi: 10.3389/fnsyn.2020.00019

[51] Shirokova OM, Sokolov RA, Pershin VI, Mukhina IV. Cultivation of primary hippocampal cell cultures for the functional and morphological matching organization of single neurons. Opera Medica at Physiologia. 2020;6(1):27–32.

[52] Shirokova O.M., Sokolov R.A., Pershin V.I., Mukhina I.V. Cultivation of primary hippocampal cell cultures for the functional and morphological matching organization of single neurons // Opera Medica et Physiologia. 2020. Vol. 6, N 1. P. 27–32.

[53] Facucho-Oliveira JM, St John JC. The relationship between pluripotency and mitochondrial DNA proliferation during early embryo development and embryonic stem cell differentiation. Stem Cell Rev Rep. 2009;5(2):140–158. doi: 10.1007/s12015-009-9058-0

[54] Facucho-Oliveira J.M., John J.C. The relationship between pluripotency and mitochondrial DNA proliferation during early embryo development and embryonic stem cell differentiation // Stem Cell Rev Rep. 2009. Vol. 5, N 2. P. 140–158. doi: 10.1007/s12015-009-9058-0

[55] Espósito MS, Piatti VC, Laplagne DA, et al. Neuronal differentiation in the adult hippocampus recapitulates embryonic development. J Neurosci. 2005;25(44):10074–10086. Corrected and republished from: J Neurosci. 2005;25:49. doi: 10.1523/JNEUROSCI.3114-05.2005

[56] Espósito M.S., Piatti V.C., Laplagne D.A., et al. Neuronal differentiation in the adult hippocampus recapitulates embryonic development // J Neurosci. 2005. Vol. 25, N 44. P. 10074–10086. Corrected and republished from: J Neurosci. 2005. Vol. 25, N 49. doi: 10.1523/JNEUROSCI.3114-05.2005

[57] Khacho M, Clark A, Svoboda DS, et al. Mitochondrial dynamics impacts stem cell identity and fate decisions by regulating a nuclear transcriptional program. Cell Stem Cell. 2016;19(2):232–247. doi: 10.1016/j.stem.2016.04.015

[58] Khacho M., Clark A., Svoboda D.S., et al. Mitochondrial dynamics impacts stem cell identity and fate decisions by regulating a nuclear transcriptional program // Cell Stem Cell. 2016. Vol. 19, N 2. P. 232–247. doi: 10.1016/j.stem.2016.04.015

[59] Zheng X, Boyer L, Jin M, et al. Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation. Elife. 2016;5:e13374. doi: 10.7554/eLife.13374

[60] Zheng X., Boyer L., Jin M., et al. Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation // Elife. 2016. Vol. 5. P. e13374. doi: 10.7554/eLife.1337

[61] Hall CN, Klein-Flügge MC, Howarth C, Attwell D. Oxidative phosphorylation, not glycolysis, powers presynaptic and postsynaptic mechanisms underlying brain information processing. J Neurosci. 2012;32(26):8940–8951. doi: 10.1523/JNEUROSCI.0026-12.2012

[62] Hall C.N., Klein-Flügge M.C., Howarth C., Attwell D. Oxidative phosphorylation, not glycolysis, powers presynaptic and postsynaptic mechanisms underlying brain information processing // J Neurosci. 2012. Vol. 32, N 26. P. 8940–8951. doi: 10.1523/JNEUROSCI.0026-12.2012

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