Neural stem cells are fundamental to development of the central nervous system (CNS)-as well as its plasticity and regeneration-and represent a potential tool for neuro transplantation therapy and research. This study is focused on examination of the proliferation dynamic and fate of embryonic neural stem cells (eNSCs) under differentiating conditions. In this work, we analyzed eNSCs differentiating alone and in the presence of sonic hedgehog (SHH) or triiodothyronine (T3) which play an important role in the development of the CNS. We found that inhibition of the SHH pathway and activation of the T3 pathway increased cellular health and survival of differentiating eNSCs. In addition, T3 was able to increase the expression of the gene for the receptor smoothened (Smo), which is part of the SHH signaling cascade, while SHH increased the expression of the T3 receptor beta gene (Thrb). This might be the reason why the combination of SHH and T3 increased the expression of the thyroxine 5-deiodinase type III gene (Dio3), which inhibits T3 activity, which in turn affects cellular health and proliferation activity of eNSCs.

Department of Biology Faculty of Medicine in Pilsen Charles University alej Svobody 1655 76 323 00 Plzen Czech Republic

Department of Histology and Embryology Faculty of Medicine in Pilsen Charles University Karlovarska 48 301 66 Plzen Czech Republic

Department of Medical Chemistry and Biochemistry Faculty of Medicine in Pilsen Charles University Karlovarska 48 301 66 Plzen Czech Republic

Department of Pathophysiology Faculty of Medicine in Pilsen Charles University alej Svobody 1655 76 323 00 Plzen Czech Republic

Laboratory of Neurodegenerative Disorders Biomedical Center Faculty of Medicine in Pilsen Charles University alej Svobody 1655 76 323 00 Plzen Czech Republic

Laboratory of Proteomics Biomedical Center Faculty of Medicine in Pilsen Charles University alej Svobody 1655 76 323 00 Plzen Czech Republic

Laboratory of Tumor Biology and Immunotherapy Biomedical Center Faculty of Medicine in Pilsen Charles University alej Svobody 1655 76 323 00 Plzen Czech Republic

Zobrazit více v PubMed

Rossi F., Cattaneo E. Opinion: Neural stem cell therapy for neurological diseases: Dreams and reality. Nat. Rev. Neurosci. 2002;3:401–409. doi: 10.1038/nrn809. PubMed DOI

Cendelin J., Buffo A., Hirai H., Magrassi L., Mitoma H., Sherrard R., Vozeh F., Manto M. Task Force Paper On Cerebellar Transplantation: Are We Ready to Treat Cerebellar Disorders with Cell Therapy? Cerebellum. 2019;18:575–592. doi: 10.1007/s12311-018-0999-1. PubMed DOI

Babuska V., Houdek Z., Tuma J., Purkartova Z., Tumova J., Kralickova M., Vozeh F., Cendelin J. Transplantation of Embryonic Cerebellar Grafts Improves Gait Parameters in Ataxic Lurcher Mice. Cerebellum. 2015;14:632–641. doi: 10.1007/s12311-015-0656-x. PubMed DOI

Dentice M., Luongo C., Huang S., Ambrosio R., Elefante A., Mirebeau-Prunier D., Zavacki A.M., Fenzi G., Grachtchouk M., Hutchin M., et al. Sonic hedgehog-induced type 3 deiodinase blocks thyroid hormone action enhancing proliferation of normal and malignant keratinocytes. Proc. Natl. Acad. Sci. USA. 2007;104:14466–14471. doi: 10.1073/pnas.0706754104. PubMed DOI PMC

Hasebe M., Ohta E., Imagawa T., Uehara M. Expression of sonic hedgehog regulates morphological changes of rat developing cerebellum in hypothyroidism. J. Toxicol. Sci. 2008;33:473–477. doi: 10.2131/jts.33.473. PubMed DOI

Desouza L.A., Sathanoori M., Kapoor R., Rajadhyaksha N., Gonzalez L.E., Kottmann A.H., Tole S., Vaidya V.A. Thyroid hormone regulates the expression of the sonic hedgehog signaling pathway in the embryonic and adult Mammalian brain. Endocrinology. 2011;152:1989–2000. doi: 10.1210/en.2010-1396. PubMed DOI PMC

Wang Y., Wang Y., Dong J., Wei W., Song B.B., Min H., Yu Y., Lei X.B., Zhao M., Teng W.P., et al. Developmental Hypothyroxinemia and Hypothyroidism Reduce Proliferation of Cerebellar Granule Neuron Precursors in Rat Offspring by Downregulation of the Sonic Hedgehog Signaling Pathway. Mol. Neurobiol. 2014;49:1143–1152. doi: 10.1007/s12035-013-8587-3. PubMed DOI

Chen Z.P., Hetzel B.S. Cretinism revisited. Best Pract. Res. Clin. Endocrinol. Metab. 2010;24:39–50. doi: 10.1016/j.beem.2009.08.014. PubMed DOI

Lindholm D., Castrén E., Tsoulfas P., Kolbeck R., Berzaghi M.D., Leingartner A., Heisenberg C.P., Tesarollo L., Parada L.F., Thoenen H. Neurotrophin-3 induced by tri-iodothyronine in cerebellar granule cells promotes Purkinje cell differentiation. J. Cell Biol. 1993;122:443–450. doi: 10.1083/jcb.122.2.443. PubMed DOI PMC

Alvarez-Dolado M., González-Sancho J.M., Bernal J., Muñoz A. Developmental expression of the tenascin-C is altered by hypothyroidism in the rat brain. Neuroscience. 1998;84:309–322. doi: 10.1016/S0306-4522(97)00511-3. PubMed DOI

Pathak A., Sinha R.A., Mohan V., Mitra K., Godbole M.M. Maternal thyroid hormone before the onset of fetal thyroid function regulates reelin and downstream signaling cascade affecting neocortical neuronal migration. Cereb. Cortex. 2011;21:11–21. doi: 10.1093/cercor/bhq052. PubMed DOI

Chen C., Zhou Z., Zhong M., Zhang Y.W., Li M.Q., Zhang L., Qu M.Y., Yang J., Wang Y., Yu Z.P. Thyroid Hormone Promotes Neuronal Differentiation of Embryonic Neural Stem Cells by Inhibiting STAT3 Signaling Through TRα1. Stem Cells Dev. 2012;21:2667–2681. doi: 10.1089/scd.2012.0023. PubMed DOI PMC

Mohan V., Sinha R.A., Pathak A., Rastogi L., Kumar P., Pal A., Godbole M.M. Maternal thyroid hormone deficiency affects the fetal neocorticogenesis by reducing the proliferating pool, rate of neurogenesis and indirect neurogenesis. Exp. Neurol. 2012;237:477–488. doi: 10.1016/j.expneurol.2012.07.019. PubMed DOI

Shimokawa N., Yousefi B., Morioka S., Yamaguchi S., Ohsawa A., Hayashi H., Azuma A., Mizuno H., Kasagi M., Masuda H., et al. Altered cerebellum development and dopamine distribution in a rat genetic model with congenital hypothyroidism. J. Neuroendocrinol. 2014;26:164–175. doi: 10.1111/jne.12135. PubMed DOI

Gothié J.D., Vancamp P., Demeneix B., Remaud S. Thyroid hormone regulation of neural stem cell fate: From development to ageing. Acta Physiol. 2020;228:e13316. doi: 10.1111/apha.13316. PubMed DOI PMC

Bauer M., Heinz A., Whybrow P.C. Thyroid hormones, serotonin and mood: Of synergy and significance in the adult brain. Mol. Psychiatry. 2002;7:140–156. doi: 10.1038/sj.mp.4000963. PubMed DOI

Billon N., Jolicoeur C., Tokumoto Y., Vennstrom B., Raff M. Normal timing of oligodendrocyte development depends on thyroid hormone receptor alpha 1 (TRalpha1) EMBO J. 2002;21:6452–6460. doi: 10.1093/emboj/cdf662. PubMed DOI PMC

Jones S.A., Jolson D.M., Cuta K.K., Mariash C.N., Anderson G.W. Triiodothyronine is a survival factor for developing oligodendrocytes. Mol. Cell. Endocrinol. 2003;199:49–60. doi: 10.1016/S0303-7207(02)00296-4. PubMed DOI

Ambrogini P., Cuppini R., Ferri P., Mancini C., Ciaroni S., Voci A., Gerdoni E., Gallo G. Thyroid hormones affect neurogenesis in the dentate gyrus of adult rat. Neuroendocrinology. 2005;81:244–253. doi: 10.1159/000087648. PubMed DOI

Desouza L.A., Ladiwala U., Daniel S.M., Agashe S., Vaidya R.A., Vaidya V.A. Thyroid hormone regulates hippocampal neurogenesis in the adult rat brain. Mol. Cell. Neurosci. 2005;29:414–426. doi: 10.1016/j.mcn.2005.03.010. PubMed DOI

Li J., Abe K., Milanesi A., Liu Y.Y., Brent G.A. Thyroid Hormone Protects Primary Cortical Neurons Exposed to Hypoxia by Reducing DNA Methylation and Apoptosis. Endocrinology. 2019;160:2243–2256. doi: 10.1210/en.2019-00125. PubMed DOI

Ingham P.W., McMahon A.P. Hedgehog signaling in animal development: Paradigms and principles. Genes Dev. 2001;15:3059–3087. doi: 10.1101/gad.938601. PubMed DOI

Wallace V.A. Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum. Curr. Biol. 1999;9:445–448. doi: 10.1016/S0960-9822(99)80195-X. PubMed DOI

Machold R., Hayashi S., Rutlin M., Muzumdar M.D., Nery S., Corbin J.G., Gritli-Linde A., Dellovade T., Porter J.A., Rubin L.L., et al. Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches. Neuron. 2003;39:937–950. doi: 10.1016/S0896-6273(03)00561-0. PubMed DOI

Lai K., Kaspar B.K., Gage F.H., Schaffer D.V. Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo. Nat. Neurosci. 2003;6:21–27. doi: 10.1038/nn983. PubMed DOI

Cai C., Thorne J., Grabel L. Hedgehog Serves as a Mitogen and Survival Factor During Embryonic Stem Cell Neurogenesis. Stem Cells. 2008;26:1097–1108. doi: 10.1634/stemcells.2007-0684. PubMed DOI

Jiao J., Chen D.F. Induction of Neurogenesis in Nonconventional Neurogenic Regions of the Adult Central Nervous System by Niche Astrocyte-Produced Signals. Stem Cells. 2008;26:1221–1230. doi: 10.1634/stemcells.2007-0513. PubMed DOI PMC

Bidet M., Joubert O., Lacombe B., Ciantar M., Nehme R., Mollat P., Bretillon L., Faure H., Bittman R., Ruat M., et al. The Hedgehog Receptor Patched Is Involved in Cholesterol Transport. PLoS ONE. 2011;6:e23834. doi: 10.1371/journal.pone.0023834. PubMed DOI PMC

Amakye D., Jagani Z., Dorsch M. Unraveling the therapeutic potential of the Hedgehog pathway in cancer. Nat. Med. 2013;19:1410–1422. doi: 10.1038/nm.3389. PubMed DOI

Bianco A.C., Salvatore D., Gereben B., Berry M.J., Larsen P.R. Biochemistry, cellular and molecular biology, and physiological roles of the iodothyronine selenodeiodinases. Endocr. Rev. 2002;23:38–89. doi: 10.1210/edrv.23.1.0455. PubMed DOI

Luongo C., Ambrosio R., Salzano S., Dlugosz A.A., Missero C., Dentice M. The sonic hedgehog-induced type 3 deiodinase facilitates tumorigenesis of basal cell carcinoma by reducing Gli2 inactivation. Endocrinology. 2014;155:2077–2088. doi: 10.1210/en.2013-2108. PubMed DOI PMC

Rohner A., Spilker M.E., Lam J.L., Pascual B., Bartkowski D., Li Q.J., Yang A.H., Stevens G., Xu M.R., Wells P.A., et al. Effective targeting of Hedgehog signaling in a medulloblastoma model with PF-5274857, a potent and selective Smoothened antagonist that penetrates the blood-brain barrier. Mol. Cancer Ther. 2012;11:57–65. doi: 10.1158/1535-7163.MCT-11-0691. PubMed DOI

Barltrop J.A., Owen T.C., Cory A.H., Cory J.G. 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl)tetrazolium, inner salt (MTS) and related analogs of 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) reducing to purple water-soluble formazans As cell-viability indicators. Bioorg. Med. Chem. Lett. 1991;1:611–614. doi: 10.1016/S0960-894X(01)81162-8. DOI

Liu Y., Peterson D.A., Kimura H., Schubert D. Mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. J. Neurochem. 1997;69:581–593. doi: 10.1046/j.1471-4159.1997.69020581.x. PubMed DOI

Malhotra A., Dey A., Prasad N., Kenney A.M. Sonic Hedgehog Signaling Drives Mitochondrial Fragmentation by Suppressing Mitofusins in Cerebellar Granule Neuron Precursors and Medulloblastoma. Mol. Cancer Res. 2016;14:114–124. doi: 10.1158/1541-7786.MCR-15-0278. PubMed DOI PMC

Casas F., Rochard P., Rodier A., Cassar-Malek I., Marchal-Victorion S., Wiesner R.J., Cabello G., Wrutniak C. A variant form of the nuclear triiodothyronine receptor c-ErbAα1 plays a direct role in regulation of mitochondrial RNA synthesis. Mol. Cell. Biol. 1999;19:7913–7924. doi: 10.1128/MCB.19.12.7913. PubMed DOI PMC

Albrecht P.J., Dahl J.P., Stoltzfus O.K., Levenson R., Levison S.W. Ciliary neurotrophic factor activates spinal cord astrocytes, stimulating their production and release of fibroblast growth factor-2, to increase motor neuron survival. Exp. Neurol. 2002;173:46–62. doi: 10.1006/exnr.2001.7834. PubMed DOI

Ding Q., Motoyama J., Gasca S., Mo R., Sasaki H., Rossant J., Hui C.C. Diminished Sonic hedgehog signaling and lack of floor plate differentiation in Gli2 mutant mice. Dev. Camb. Engl. 1998;125:2533–2543. PubMed

Sheng H., Goich S., Wang A.Q., Grachtchouk M., Lowe L., Mo R., Lin K., de Sauvage F.J., Sasaki H., Hui C.C., et al. Dissecting the oncogenic potential of Gli2: Deletion of an NH2-terminal fragment alters skin tumor phenotype. Cancer Res. 2002;62:5308–5316. PubMed

Aw D.K.L., Sinha R.A., Tan H.C., Loh L.M., Salvatore D., Yen P.M. Studies of molecular mechanisms associated with increased deiodinase 3 expression in a case of consumptive hypothyroidism. J. Clin. Endocrinol. Metab. 2014;99:3965–3971. doi: 10.1210/jc.2013-3408. PubMed DOI

Gil-Ibáñez P., Bernal J., Morte B. Thyroid Hormone Regulation of Gene Expression in Primary Cerebrocortical Cells: Role of Thyroid Hormone Receptor Subtypes and Interactions with Retinoic Acid and Glucocorticoids. PLoS ONE. 2014;9:e91692. doi: 10.1371/journal.pone.0091692. PubMed DOI PMC

Kapoor R., Ghosh H., Nordstrom K., Vennstrom B., Vaidya V.A. Loss of thyroid hormone receptor β is associated with increased progenitor proliferation and NeuroD positive cell number in the adult hippocampus. Neurosci. Lett. 2011;487:199–203. doi: 10.1016/j.neulet.2010.10.022. PubMed DOI PMC

Portella A.C., Carvalho F., Faustino L., Wondisford F.E., Ortiga-Carvalho T.M., Comes F.C.A. Thyroid hormone receptor beta mutation causes severe impairment of cerebellar development. Mol. Cell. Neurosci. 2011;44:68–77. doi: 10.1016/j.mcn.2010.02.004. PubMed DOI

Baxi E.G., Schott J.T., Fairchild A.N., Kirby L.A., Karani R., Uapinyoying P., Pardo-Villamizar C., Rothstein J.R., Bergles D.E., Calabresi P.A. A selective thyroid hormone β receptor agonist enhances human and rodent oligodendrocyte differentiation. Glia. 2014;62:1513–1529. doi: 10.1002/glia.22697. PubMed DOI PMC

Park J.W., Zhao L., Willingham M., Cheng S.Y. Oncogenic mutations of thyroid hormone receptor β. Oncotarget. 2015;6:8115–8131. doi: 10.18632/oncotarget.3466. PubMed DOI PMC

Joseph B., Ji M., Liu D., Hou P., Xing M.Z. Lack of mutations in the thyroid hormone receptor (TR) alpha and beta genes but frequent hypermethylation of the TR beta gene in differentiated thyroid tumors. J. Clin. Endocrinol. Metab. 2007;92:4766–4770. doi: 10.1210/jc.2007-0812. PubMed DOI

Furuya S., Makino A., Hirabayashi Y. An improved method for culturing cerebellar Purkinje cells with differentiated dendrites under a mixed monolayer setting. Brain Res. Protoc. 1998;3:192–198. doi: 10.1016/S1385-299X(98)00040-3. PubMed DOI

Leong C., Zhai D., Kim B., Yun S.W., Chang Y.T. Neural stem cell isolation from the whole mouse brain using the novel FABP7-binding fluorescent dye, CDr3. Stem Cell Res. 2013;11:1314–1322. doi: 10.1016/j.scr.2013.09.002. PubMed DOI

Cooper-Kuhn C.M., Kuhn H.G. Is it all DNA repair? Methodological considerations for detecting neurogenesis in the adult brain. Dev. Brain Res. 2002;134:13–21. doi: 10.1016/S0165-3806(01)00243-7. PubMed DOI

Kempermann G., Gast D., Kronenberg G., Yamaguchi M., Gage F.H. Early determination and long-term persistence of adult-generated new neurons in the hippocampus of mice. Dev. Camb. Engl. 2003;130:391–399. doi: 10.1242/dev.00203. PubMed DOI

Sundberg M., Savola S., Hienola A., Korhonen L., Lindholm D. Glucocorticoid hormones decrease proliferation of embryonic neural stem cells through ubiquitin-mediated degradation of cyclin D1. J. Neurosci. 2006;26:5402–5410. doi: 10.1523/JNEUROSCI.4906-05.2006. PubMed DOI PMC

Kamentsky L., Jones T.R., Fraser A., Bray M.A., Logan D.J., Madden K.L., Ljosa V., Rueden C., Eliceiri K.W., Carpenter A.E. Improved structure, function and compatibility for CellProfiler: Modular high-throughput image analysis software. Bioinformatics. 2011;27:1179–1180. doi: 10.1093/bioinformatics/btr095. PubMed DOI PMC

Agger S.A., Marney L.C., Hoofnagle A.N. Simultaneous Quantification of Apolipoprotein A-I and Apolipoprotein B by Liquid-Chromatography-Multiple-Reaction-Monitoring Mass Spectrometry. Clin. Chem. 2010;56:1804–1813. doi: 10.1373/clinchem.2010.152264. PubMed DOI PMC

Manly B.F.J. Randomization, Bootstrap, and Monte Carlo Methods in Biology. 3rd ed. Chapman & Hall/ CRC; Boca Raton, FL, USA: 2007.

Benjamini Y., Hochberg Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J. R. Stat. Soc. Ser. B Methodol. 1995;57:289–300. doi: 10.1111/j.2517-6161.1995.tb02031.x. DOI