Kim Sanghoon, Pajarillo Edward, Digman Alexis, Ajayi Itunu, Son Deok-Soo, Aschner Michael, Lee Eunsook
Department of Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL, USA.
Department of Biochemistry, Cancer Biology, Neuroscience, and Pharmacology, Meharry Medical College, Nashville, TN, USA.
Neurochem Res. 2025 Jul 16;50(4):233. doi: 10.1007/s11064-025-04474-z.
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.
脑多巴胺能（DAergic）神经元在介导运动、奖赏和认知过程中起关键作用，但特别容易受到与帕金森病和锰中毒相关的毒性损伤。几种细胞系已被用于研究多巴胺能功能和毒性，每种细胞系都有独特的优缺点。在这里，我们研究了源自小鼠的儿茶酚胺能细胞系——阴极α-分化（CAD）细胞是否适合作为体外模型用于多巴胺能神经毒性研究，重点关注其合成多巴胺（DA）的能力以及关键相关蛋白的表达。还测试了锰（Mn）以确定其多巴胺能毒性潜力。通过血清剥夺诱导CAD细胞分化。使用高效液相色谱、蛋白质免疫印迹和逆转录定量聚合酶链反应来评估多巴胺水平以及多巴胺能蛋白如酪氨酸羟化酶（TH）、芳香族L-氨基酸脱羧酶（AAAD）、囊泡单胺转运体2（VMAT-2）和多巴胺转运体（DAT）的表达。结果表明，与未分化细胞相比，分化后的CAD细胞具有更高的多巴胺水平。多巴胺前体L-3,4-二羟基苯丙氨酸（L-DOPA）增加了多巴胺的产生，而AAAD抑制剂卡比多巴则降低了其产量。CAD细胞还表达AAAD蛋白，表明后者参与该细胞系中的多巴胺合成。此外，锰降低了多巴胺以及多巴胺合成酶如TH和AAAD以及VMAT-2的mRNA和蛋白水平，从而损害了多巴胺能系统。综上所述，分化后的CAD细胞具有合成多巴胺和表达多巴胺合成酶的能力。此外，锰在CAD细胞中引起了多巴胺能毒性，这表明CAD细胞适合用于研究多巴胺能神经毒性。
