Immortalized Rat Neuronal Cell Line Shows Potential as an Improved Cell Model for Dopamine Transporter Research

Abstract

Dopamine (DA) is a neurotransmitter which controls several psychological and physiological processes such as reward, motivation, attention, and motor activity. Disruption of DA homeostasis 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. In addition, 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. Thus, a greater understanding of DAT functional and regulatory 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 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 very 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 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), DOPA decarboxylase (DOPA‐DC), and native DAT via immunoblotting, finding modest TH and DOPA‐DC and very low DAT protein levels. Differentiation 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 other proteins important for neuron physiology such as syntaxin 1a, vesicular monoamine transporter 2 (VMAT2), dopamine receptor D2 (D2R), and glutamate receptors (AMPA, mGluR, and NMDA). 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, indicating no detectable DAT or norepinephrine transporter expression. These stable transformants also showed amphetamine‐stimulated DA efflux, a characteristic, pharmacological response in dopaminergic neurons. Using an ELISA specific for detecting DA, similar extracellular DA levels were detected in the rDAT N27 line compared to the parental N27 line, demonstrating DA production in these lines. To support our ELISA results, we have begun to assess N27 cells for DA production using a highly sensitive liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) assay. Taken together, our results suggest that these cells may serve as suitable neuronal models in investigating DAT function and regulation. However, further studies are required to confirm this assertion.Support or Funding InformationSupported by the National Institute on Drug Abuse Grant DA 031991 JDF, P20 RR017699 (to U.N.D.) from the COBRE program of the National Center for Research Resources, and P20 RR016741 (to U.N.D.) from the INBRE program of the National Center for Research Resources.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.