Assessment of an Immortalized Rat Dopaminergic Neuronal Cell Line as a Potential Model for Studying Dopamine Transporter Function
Authors: Larson Garret H , Stanislowski Daniel J , Foster James D 
Published: November 2023

Dopamine (DA) is a neurotransmitter which controls several psychological and physiological processes including, but not limited to, reward, motivation, attention, and motor activity. Disruption of DA homeostasis in the brain is implicated in several neurological diseases such as attention‐deficit hyperactivity disorder (ADHD), schizophrenia, depression, and Parkinson disease. The DA transporter (DAT) is an integral plasma membrane protein that actively translocates extracellular DA into the presynaptic neuron. DAT serves as a primary mechanism by which synaptic DA levels and neurotransmission is spatially and temporally modulated. DAT is a major site of action for the psychostimulants cocaine and amphetamine, and for therapeutic drugs such as methylphenidate and bupropion used to treat ADHD and depression. Because DAT function is crucial for DA homeostasis, the protein is posited to play an important role in the etiology of DA imbalance diseases. Thus, a greater understanding of DAT functional and regulatory molecular mechanisms is critical as this may lead to additional effective therapeutic interventions. Currently, many cell models are used in exploring DAT function and regulation. However, it is uncertain if any of these cell lines adequately replicate dopaminergic neuron physiology. Primary dopaminergic neuron cultures from brain tissue appear to provide the most direct and reliable model for studying dopaminergic functions but are removed from their specialized neuronal environment and limited in number, restricting their usefulness for many biochemical assays. In addition, primary cultures are labor intensive and challenging to prepare and maintain. To address these issues, we are currently examining the feasibility of the neuronal cell line, 1RB3AN27 (N27), derived from immortalized rat primary dopaminergic neurons as an improved cell model for studying DAT. We have assessed these cells for expression of key dopaminergic proteins such as tyrosine hydroxylase (TH), syntaxin 1A, and native DAT via immunoblotting, finding modest TH and syntaxin 1A and very low DAT protein levels. Differentiation in the presence of dibutyryl cAMP and dehydroepiandrosterone failed to elevate protein levels of these dopaminergic markers, although more experimental replicates are needed to confirm these observations. We are currently determining the levels of additional proteins involved in DAT regulation and DA biosynthesis such as palmitoyl acyltransferases (PATs), DOPA decarboxylase, and Nurr1. Due to very low DAT levels in both undifferentiated and differentiated cells, we transfected these cells with human (h) or rat (r) DAT, creating lines stably expressing these DAT forms. Both the rDAT and hDAT N27 lines showed robust DA uptake capacity compared to the native N27 line in which little to no DA uptake was detected. In an initial kinetic analysis, the Km DA determined for the hDAT N27 line was lower than that determined using other heterologous cell systems but was similar to that determined using rat striatal synaptosomes. Taken together, preliminary results suggest that these cell lines may serve as suitable neuronal models in investigating DAT function and regulation. However, further studies are required to make this determination.Support or Funding InformationNational Institutes of Health grants DA 031991 (JDF), DA13147 and 5P20‐104360 (RAV), P30‐GM103329 (UND), P20‐GM12345 (UND)