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Cholinergic Differentiation of Human Neuroblastoma SH-SY5Y Cell Line and Its Potential Use as an In vitro Model for Alzheimer’s Disease Studies

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Abstract

Cholinergic transmission is critical to high-order brain functions such as memory, learning, and attention. Alzheimer’s disease (AD) is characterized by cognitive decline associated with a specific degeneration of cholinergic neurons. No effective treatment to prevent or reverse the symptoms is known. Part of this might be due to the lack of in vitro models that effectively mimic the relevant features of AD. Here, we describe the characterization of an AD in vitro model using the SH-SY5Y cell line. Exponentially growing cells were maintained in DMEM/F12 medium and differentiation was triggered by the combination of retinoic acid (RA) and BDNF. Both acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) enzymatic activities and immunocontent were determined. For mimicking tau and amyloid-β pathology, RA + BDNF-differentiated cells were challenged with okadaic acid (OA) or soluble oligomers of amyloid-β (AβOs) and neurotoxicity was evaluated. RA + BDNF-induced differentiation resulted in remarkable neuronal morphology alterations characterized by increased neurite density. Enhanced expression and enzymatic activities of cholinergic markers were observed compared to RA-differentiation only. Combination of sublethal doses of AβOs and OA resulted in decreased neurite densities, an in vitro marker of synaptopathy. Challenging RA + BDNF-differentiated SH-SY5Y cells with the combination of sublethal doses of OA and AβO, without causing considerable decrease of cell viability, provides an in vitro model which mimics the early-stage pathophysiology of cholinergic neurons affected by AD.

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Abbreviations

AD:

Alzheimer’s disease

DMEM:

Dulbecco’s Modified Eagle Medium

FBS:

Fetal bovine serum

RA:

Retinoic acid

BDNF:

Brain-derived neurotrophic factor

AChE:

Acetylcholinesterase

ChAT:

Choline acetyltransferase

AβOs:

Amyloid-β oligomers

AO:

Okadaic acid

MTT:

3-(4,5-Dimethylthiazol-2il)-2,5-diphenyltetrazolium bromide

DAT:

Dopamine transporter

Aβ:

Amyloid-β

BFC:

Basal forebrain complex

APP:

Amyloid precursor protein

ACh:

Acetylcholine

CDK5:

Cyclin-dependent kinase

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Funding

This study was supported by the Brazilian funds MCTI/CNPq INCT-TM/CAPES/FAPESP (465458/2014-9), CNPq/MS/SCTIE/DECIT - Pesquisas Sobre Doenças Neurodegenerativas (466989/2014-8), and PRONEX/FAPERGS (16/2551-0000499-4). FK received a fellowship from MCT/CNPq [306439/2014-0]. ERZ receives financial support from CAPES [88881.141186/2017-01].

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Authors and Affiliations

  1. Laboratory of Cellular Biochemistry, Department of Biochemistry, ICBS/UFRGS, 2600 Ramiro Barcelos St, Porto Alegre, RS, 90035-003, Brazil

    Liana M. de Medeiros, Marco A. De Bastiani, Patrícia Schonhofen, Bianca Pfaffenseller, Bianca Wollenhaupt-Aguiar & Fábio Klamt

  2. National Institutes of Science and Technology–Translational Medicine (INCT-TM), Porto Alegre, Brazil

    Liana M. de Medeiros, Marco A. De Bastiani, Patrícia Schonhofen, Bianca Pfaffenseller, Bianca Wollenhaupt-Aguiar & Fábio Klamt

  3. Post-Graduate Program in Biochemistry, Department of Biochemistry, ICBS/UFRGS, Porto Alegre, RS, 90035-003, Brazil

    Liana M. de Medeiros, Marco A. De Bastiani, Patrícia Schonhofen, Lucas Grun, Florência Barbé-Tuana, Eduardo R. Zimmer & Fábio Klamt

  4. Programa de Pós-Graduação em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense (UNESC), Criciúma, SC, 88806-000, Brazil

    Eduardo P. Rico

  5. Department of Psychiatry and Behavioral Neurosciences, McMaster University, Hamilton, ON, Canada

    Bianca Pfaffenseller & Bianca Wollenhaupt-Aguiar

  6. Laboratory of Molecular Biology and Bioinformatics, Department of Biochemistry, ICBS/UFRGS, Porto Alegre, RS, 90035-003, Brazil

    Lucas Grun & Florência Barbé-Tuana

  7. Brain Institute of Rio Grande do Sul (BraIns), Pontifical Catholic University of Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil

    Eduardo R. Zimmer

  8. Department of Pharmacology, ICBS/UFRGS, Porto Alegre, RS, Brazil

    Eduardo R. Zimmer

  9. Bioinformatics and Systems Biology Laboratory, Polytechnic Center, Federal University of Paraná (UFPR), Curitiba, PR, 81520-260, Brazil

    Mauro A. A. Castro

  10. School of Cancer and Pharmaceutical Sciences, King’s College London, 150 Stamford Street, London, SE1 9NH, UK

    Richard B. Parsons

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  1. Liana M. de Medeiros
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  2. Marco A. De Bastiani
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  12. Fábio Klamt
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Correspondence to Fábio Klamt.

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de Medeiros, L.M., De Bastiani, M.A., Rico, E.P. et al. 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 (2019). https://doi.org/10.1007/s12035-019-1605-3

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