Abstract
The Cath.a-differentiated (CAD) cell line is a central nervous system-derived catecholaminergic cell line originating from tyrosine hydroxylase (TH)-producing neurons located around the locus coeruleus area of the mouse brain. CAD cells have been used as an in vitro model for cellular and molecular studies due to their ability to differentiate under serum-free media conditions. However, the lack of serum-derived survival factors, limits the longevity for differentiated CAD cells to be maintained in healthy conditions; thereby, limiting their use in long-term culture studies. Here, we present a novel differentiation method that utilizes dexamethasone (Dex), a synthetic glucocorticoid receptor agonist. Specifically, we discovered that the addition of 100 µM of Dex into the 1% fetal bovine serum (FBS)-supplemented media effectively induced neuronal differentiation of CAD cells, as characterized by neurite formation and elongation. Dex-differentiated CAD cells exited the cell cycle, stopped proliferating, extended the neurites, and expressed neuronal markers. These effects were dependent on the glucocorticoid receptors (GR) as they were abolished by GR knockdown. Importantly, Dex-differentiated CAD cells showed longer survival duration than serum-free differentiated CAD cells. In addition, RNA-sequencing and qPCR data demonstrate that several genes involved in proliferation, neuronal differentiation, and survival pathways were differentially expressed in the Dex-differentiated cells. This is the first study to reveal Dex as a novel differentiation methodology used to generate postmitotic neuronal CAD cells, which may be utilized as an in vitro neuronal model for cellular and molecular neurobiology research.
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Data Availability
All data generated or analysed during this study are included in this published article (and its supplementary information files).
Code Availability
GEO accession numbers: GSE162087.
Change history
16 June 2021
A Correction to this paper has been published: https://doi.org/10.1007/s10571-021-01119-x
Abbreviations
- BP:
-
Biological processes
- BrdU:
-
Bromodeoxyuridine
- BSA:
-
Bovine serum albumin
- CAD:
-
Cath.a-differentiated cells
- CC:
-
Cellular components
- CCNE2:
-
Cyclin E2
- CDK2:
-
Cyclin-dependent kinase 2
- CEBPB:
-
CCAAT/enhancer-binding protein beta
- CNS:
-
Central nervous system
- DAVID:
-
Database for Annotation, Visualization and Integrated Discovery
- DEGs:
-
Differentially expressed genes
- DexL:
-
Dexamethasone
- DMEM/F12:
-
Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12
- DMSO:
-
Dimethysulfoxide
- ECACC:
-
European Collection of Authenticated Cell Cultures
- E2F2:
-
E2F transcription factor 2
- FBS:
-
Fetal Bovine serum
- FDR:
-
False discovery rate
- FGF1:
-
Fibroblast growth factor-1
- FKBP5:
-
FK506 binding protein 5
- GADD45b:
-
Growth arrest and DNA damage inducible beta
- GAP-43:
-
Growth-associated protein-43
- GAPDH:
-
Glyceraldehyde 3-phosphate dehydrogenase
- GO:
-
Gene ontology
- GR:
-
Glucocorticoid receptor
- GSTA3:
-
Glutathione S-transferase A3
- HCN1:
-
Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 1
- IL6:
-
Interleukin-6
- KCNT1:
-
Potassium channel subfamily T, member 1
- MAP2:
-
Microtubule-associated protein type 2
- MAPT:
-
Microtubule-associated protein tau
- KEGG:
-
Kyoto Encyclopedia of Genes and Genomes
- MF:
-
Molecular functions
- MTT:
-
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for
- NDRG2:
-
N-myc downstream-regulated gene 2
- NGF:
-
Nerve growth factor
- NPCs:
-
Neural progenitor cells
- PBS:
-
Phosphate buffer saline
- PI:
-
Propidium iodide
- PI3K-Akt:
-
Phosphatidylinositol 3 kinase-protein kinase B
- PPAR:
-
Peroxisome proliferator-activated receptor
- PPARA:
-
Peroxisome Proliferator Activated Receptor Alpha
- Pro:
-
Proliferative condition
- qPCR:
-
Quantitative real-time polymerase chain reaction
- RRIDs:
-
Research resource identifiers
- RNA-seq:
-
RNA-sequencing
- SCN1A:
-
Sodium voltage-gated channel alpha subunit 1
- SDS-PAGE:
-
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
- SF:
-
Serum-free differentiated condition
- SYP:
-
Synaptophysin
- SYT2:
-
Synaptotagmin 2
- TBST:
-
Tris buffer saline with tween 20
- TH:
-
Tyrosine hydroxylase
- TUBB3:
-
Tubulin beta-3 class lll
- °C:
-
Degree Celsius
- CO2 :
-
Carbon dioxide
- h:
-
Hours
- mg:
-
Milligrams
- ml:
-
Milliliter
- n:
-
Number
- pM:
-
Picomolar
- v/v:
-
Volume/volume
- µg:
-
Microgram
- µL:
-
Microliter
- µm:
-
Micrometer
- µM:
-
Micromolar
- %:
-
Percentage
- i.e.,:
-
In other words
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Acknowledgements
This research project is supported by Mahidol University (NDFR20/2563), and partially by the Thailand Research Fund (IRN58W0004), and the Central Instrument Facility (CIF) grant from the Faculty of Science, Mahidol University to WS. The work was also supported by the Young Researcher Development Program from National Research Council of Thailand (NRCT), and The Science Achievement Scholarship of Thailand (SAST) to EK.
Funding
This research project is supported by Mahidol University (NDFR20/2563), and partially by the Thailand Research Fund (IRN58W0004), and the Central Instrument Facility (CIF) grant from the Faculty of Science, Mahidol University to WS. The work was also supported by the Young Researcher Development Program from National Research Council of Thailand (NRCT), and The Science Achievement Scholarship of Thailand (SAST) to EK.
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WS and EK designed the experiments. EK performed the research with the aid of KU, NB and KB, WS and EK wrote the manuscript with help from NB and KB All the authors read and approved the manuscript.
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Khongkla, E., Uppakara, K., Boonmuen, N. et al. A Novel Methodology Using Dexamethasone to Induce Neuronal Differentiation in the CNS-Derived Catecholaminergic CAD Cells. Cell Mol Neurobiol 42, 2337–2353 (2022). https://doi.org/10.1007/s10571-021-01109-z
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DOI: https://doi.org/10.1007/s10571-021-01109-z