A Novel Methodology Using Dexamethasone to Induce Neuronal Differentiation in the CNS-Derived Catecholaminergic CAD Cells

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. Speci cally, 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. Dexdifferentiated 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 rst 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.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 bromidefor, NDRG2 = N-myc downstream-