Abstract
Pathological alterations of the neuronal Tau protein are characteristic for many neurodegenerative diseases, called tauopathies. To investigate the underlying mechanisms of tauopathies, human neuronal cell models are required to study Tau physiology and pathology in vitro. Primary rodent neurons are an often used model for studying Tau, but rodent Tau differs in sequence, splicing, and aggregation propensity, and rodent neuronal physiology cannot be compared to humans. Human-induced pluripotent stem cell (hiPSC)-derived neurons are expensive and time-consuming. Therefore, the human neuroblastoma SH-SY5Y cell line is a commonly used cell model in neuroscience as it combines convenient handling and low costs with the advantages of human-derived cells. Since naïve SH-SY5Y cells show little similarity to human neurons and almost no Tau expression, differentiation is necessary to obtain human-like neurons for studying Tau protein-related aspects of health and disease. As they express in principle all six Tau isoforms seen in the human brain, differentiated SH-SY5Y-derived neurons are suitable for investigating the human microtubule-associated protein Tau and, for example, its sorting and trafficking. Here, we describe and discuss a general cultivation procedure as well as four differentiation methods to obtain SH-SY5Y-derived neurons resembling noradrenergic, dopaminergic, and cholinergic properties, based on the treatment with retinoic acid (RA), brain-derived neurotrophic factor (BDNF), and 12-O-tetrade canoylphorbol-13-acetate (TPA). TPA and RA-/TPA-based protocols achieve differentiation efficiencies of 40–50% after 9 days of treatment. The highest differentiation efficiency (~75%) is accomplished by a combination of RA and BDNF; treatment only with RA is the most time-efficient method as ~50% differentiated cells can be obtained already after 7 days.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Arendt T, Stieler JT, Holzer M (2016) Tau and tauopathies. Brain Res Bull 126:238–292. https://doi.org/10.1016/j.brainresbull.2016.08.018
Zhang X, Gao F, Wang D, Li C, Fu Y, He W, Zhang J (2018) Tau pathology in Parkinson’s disease. Front Neurol 9. https://doi.org/10.3389/fneur.2018.00809
Agholme L, Lindström T, Kgedal K, Marcusson J, Hallbeck M (2010) An in vitro model for neuroscience: differentiation of SH-SY5Y cells into cells with morphological and biochemical characteristics of mature neurons. J Alzheimers Dis 20:1069–1082. https://doi.org/10.3233/JAD-2010-091363
Bell M, Zempel H (2022) SH-SY5Y-derived neurons: a human neuronal model system for investigating TAU sorting and neuronal subtype-specific TAU vulnerability. Rev Neurosci 33:1–15. https://doi.org/10.1515/revneuro-2020-0152
Janke C, Beck M, Stahl T, Holzer M, Brauer K, Bigl V, Arendt T (1999) Phylogenetic diversity of the expression of the microtubule-associated protein tau: implications for neurodegenerative disorders. Mol Brain Res 68:119–128. https://doi.org/10.1016/S0169-328X(99)00079-0
Kovalevich J, Langford D (2013) Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology. Methods Mol Biol. https://doi.org/10.1007/978-1-62703-640-5_2
Encinas M, Iglesias M, Liu Y, Wang H, Muhaisen A, Ceña V, Gallego C, Comella JX (2000) Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells. J Neurochem 75:991–1003. https://doi.org/10.1046/j.1471-4159.2000.0750991.x
Lopes FM, Schröder R, Júnior MLC d F, Zanotto-Filho A, Müller CB, Pires AS, Meurer RT, Colpo GD, Gelain DP, Kapczinski F, Moreira JCF, Fernandes M d C, Klamt F (2010) Comparison between proliferative and neuron-like SH-SY5Y cells as an in vitro model for Parkinson disease studies. Brain Res 1337:85–94. https://doi.org/10.1016/j.brainres.2010.03.102
Bachmann S, Bell M, Klimek J, Zempel H (2021) Differential effects of the six human TAU isoforms: somatic retention of 2N-TAU and increased microtubule number induced by 4R-TAU. Front Neurosci 15. https://doi.org/10.3389/fnins.2021.643115
Schneider L, Giordano S, Zelickson BR, Johnson MS, Benavides GA, Ouyang X, Fineberg N, Darley-Usmar VM, Zhang J (2011) Differentiation of SH-SY5Y cells to a neuronal phenotype changes cellular bioenergetics and the response to oxidative stress. Free Radic Biol Med 51:2007–2017. https://doi.org/10.1016/j.freeradbiomed.2011.08.030.Differentiation
Bell M, Zempel H (2022) A simple human cell model for TAU trafficking and tauopathy-related TAU pathology. Neural Regen Res 17:770–772. https://doi.org/10.4103/1673-5374.322450
Bell M, Bachmann S, Klimek J, Langerscheidt F, Zempel H (2021) Axonal TAU sorting requires the C-terminus of TAU but is independent of ANKG and TRIM46 enrichment at the AIS. Neuroscience 461:155–171. https://doi.org/10.1016/j.neuroscience.2021.01.041
Neuhaus JFG, Baris OR, Hess S, Moser N, Schröder H, Chinta SJ, Andersen JK, Kloppenburg P, Wiesner RJ (2014) Catecholamine metabolism drives generation of mitochondrial DNA deletions in dopaminergic neurons. Brain 137:354–365. https://doi.org/10.1093/brain/awt291
de Medeiros LM, De Bastiani MA, Rico EP, Schonhofen P, Pfaffenseller B, Wollenhaupt-Aguiar B, Grun L, Barbé-Tuana F, Zimmer ER, Castro MAA, Parsons RB, Klamt F (2019) Cholinergic differentiation of human neuroblastoma SH-SY5Y cell line and its potential use as an in vitro model for Alzheimer’s disease studies. Mol Neurobiol 56:7355–7367. https://doi.org/10.1007/s12035-019-1605-3
Shipley MM, Mangold CA, Szpara ML (2016) Differentiation of the SH-SY5Y human neuroblastoma cell line. J Vis Exp 2016:1–11. https://doi.org/10.3791/53193
Magalingam KB, Radhakrishnan AK, Somanath SD, Md S, Haleagrahara N (2020) Influence of serum concentration in retinoic acid and phorbol ester induced differentiation of SH-SY5Y human neuroblastoma cell line. Mol Biol Rep 47:8775–8788. https://doi.org/10.1007/s11033-020-05925-2
Şahin M, Öncü G, Yılmaz MA, Özkan D, Saybaşılı H (2021) Transformation of SH-SY5Y cell line into neuron-like cells: investigation of electrophysiological and biomechanical changes. Neurosci Lett 745:135628. https://doi.org/10.1016/j.neulet.2021.135628
Teppola H, Sarkanen JR, Jalonen TO, Linne ML (2016) Morphological differentiation towards neuronal phenotype of SH-SY5Y neuroblastoma cells by estradiol, retinoic acid and cholesterol. Neurochem Res 41:731–747. https://doi.org/10.1007/s11064-015-1743-6
Simpson PB, Bacha JI, Palfreyman EL, Woollacott AJ, McKernan RM, Kerby J (2001) Retinoic acid-evoked differentiation of neuroblastoma cells predominates over growth factor stimulation: an automated image capture and quantitation approach to neuritogenesis. Anal Biochem 298:163–169. https://doi.org/10.1006/abio.2001.5346
Kume T, Kawato Y, Osakada F, Izumi Y, Katsuki H, Nakagawa T, Kaneko S, Niidome T, Takada-Takatori Y, Akaike A (2008) Dibutyryl cyclic AMP induces differentiation of human neuroblastoma SH-SY5Y cells into a noradrenergic phenotype. Neurosci Lett 443:199–203. https://doi.org/10.1016/j.neulet.2008.07.079
Madadi Z, Akbari-Birgani S, Mohammadi S, Khademy M, Mousavi SA (2021) The effect of caspase-9 in the differentiation of SH-SY5Y cells. Eur J Pharmacol 904:174138. https://doi.org/10.1016/j.ejphar.2021.174138
Riegerová P, Brejcha J, Bezděková D, Chum T, Mašínová E, Čermáková N, Ovsepian SV, Cebecauer M, Štefl M (2021) Expression and localization of AβPP in SH-SY5Y cells depends on differentiation state. J Alzheimers Dis 82:485–491. https://doi.org/10.3233/JAD-201409
Krishtal J, Metsla K, Bragina O, Tõugu V, Palumaa P (2019) Toxicity of amyloid-β peptides varies depending on differentiation route of SH-SY5Y cells. J Alzheimers Dis 71:879–887. https://doi.org/10.3233/JAD-190705
Acknowledgments
The authors thank Jennifer Klimek for the excellent technical assistance, Sarah Bachmann for fruitful discussions and advice, Prof. Dr. Rudi Wiesner (Institute for Veg. Physiology II, University Hospital Cologne, Cologne) for providing cells, Dr. Magdalena Bogus (Institute of Forensic Medicine, University Hospital Cologne, Cologne) for cell authentication, and Jana Chudobová for critically reading the manuscript. This research is supported by the Köln Fortune Program/Faculty of Medicine, University of Cologne, by the Else-Kröner-Fresenius-Stiftung, and by a stipend from the Deutsche Studienstiftung.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Langerscheidt, F., Bell-Simons, M., Zempel, H. (2024). Differentiating SH-SY5Y Cells into Polarized Human Neurons for Studying Endogenous and Exogenous Tau Trafficking: Four Protocols to Obtain Neurons with Noradrenergic, Dopaminergic, and Cholinergic Properties. In: Smet-Nocca, C. (eds) Tau Protein. Methods in Molecular Biology, vol 2754. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3629-9_30
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3629-9_30
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3628-2
Online ISBN: 978-1-0716-3629-9
eBook Packages: Springer Protocols