CAD (Cath. a-Differentiated) Cells Produce Dopamine along with Dopamine-Synthesizing Enzymes

Abstract

Brain dopaminergic (DAergic) neurons play a critical role in mediating motor, reward, and cognitive processes, but are particularly vulnerable to toxic insults associated with Parkinson’s disease and manganism. Several cell lines have been used to study DAergic function and toxicity, and each has distinct advantages and limitations. Here, we investigated whether cath. a-differentiated (CAD) cells, a mouse-derived catecholaminergic cell line, are suitable for DAergic neurotoxicity research as an in vitro model, focusing on their ability to synthesize dopamine (DA) and the expression of key associated proteins. Manganese (Mn) was also tested to determine its DAergic toxicity potential. CAD cells were differentiated with serum deprivation. High-performance liquid chromatography, western blotting, and RT-qPCR were used to assess DA levels, and the expressions of DAergic proteins such as tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AAAD), vesicular monoamine transporter-2 (VMAT-2), and DA transporter (DAT). The results showed that differentiated CAD cells had higher DA levels compared to undifferentiated cells. L-3,4-dihydroxyphenylalanine (L-DOPA), the DA precursor, increased DA production, while carbidopa, an AAAD inhibitor, decreased its production. CAD cells also expressed AAAD protein, indicating that the latter participates in DA synthesis in this cell line. Moreover, Mn decreased DA as well as mRNA and protein levels of DA synthesizing enzymes, such as TH and AAAD, and VMAT-2, thus impairing the DAergic system. Taken together, differentiated CAD cells possess the capability to synthesize DA and express DA-synthesizing enzymes. In addition, Mn caused DAergic toxicity in CAD cells, suggesting that CAD cells are suitable for studying DAergic neurotoxicity.