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We here report the decreased expression level of dopamine D2 receptors in human dopaminergic neurons generated from opioid-dependent iPSCs (inducible pluripotent stem cells) compared to dopaminergic neurons generated from control iPSCs and expression of kappa and delta but not mu opioid receptor subtypes in these cells.
The dopaminergic system has been implicated to play important roles in addiction. However human dopaminergic neurons from drug dependent subjects were not available for study until recent developments in iPSCs technology. Our objective was to study human dopaminergic neurons derived from drug-dependent individuals. iPSCs were derived from 4 subjects (2 control and 2 opioid-dependent subjects) as shown in Figure 1. We successfully generated midbrain DA neurons that expressed Forkhead Box A2 (FOXA2), LIM Homeobox Transcription Factor 1, Alpha (LMX1A), tyrosine hydroxylase (TH), DA D2 receptor, Nuclear receptor related 1 protein (Nurr1), vesicular monoamine transporter 2 (VMAT2) and the dopamine transporter (DAT) (Fig 1). All iPSC lines generated DA neurons that released DA detectable by HPLC (ranging from 13.30±1.62 to 60.52±2.78 nM). There was no apparent difference in DA differentiation efficiency between the cell lines derived from control and opioid dependent subjects with regards to the percentage of TH positive cells. Importantly, there was a differential expression levels of Drd2 receptors in control iPSCs-DA neurons compared to opioid dependent iPSC derived DA neurons. Specifically, our results showed lower expression levels of Drd2 dopamine receptors in both of the opioid dependent iPSC lines compared to the DA neurons derived from control iPSCs; relative DA D2 mRNA levels equal to 0.37±0.02 and 0.33±0.04 for the opioid-dependent dopaminergic neurons and 0.50±0.05 and 0.67±0.07 for control dopaminergic neurons. This is consistent with previous neuroimaging findings that DA D2 receptor levels are lower in both opioid dependent subjects and in a variety of other addictions (1, 2). We also examined the expression of different classes of opioid receptors. Previous reports in animal studies suggest that mu receptor specific opioid agonists might excite DA neurons only through indirect mechanisms (3). Our data showed that mu opioid receptors (MOR) were not detected in our differentiated human DA neurons derived from iPS cells, supporting this hypothesis in human DA neurons. In contrast, kappa and delta opioid receptor (KOR and DOR) were detected in our human DA neuronal cultures from all four iPSC lines. This is consistent with the report that kappa opioid agonist can directly inhibit midbrain DA neurons and that delta receptor activation in substantia nigra (SN) or ventral tegmantal area (VTA) results in protection in PD models and protection against alcohol addiction (4, 5). Although iPSCs have been used in multiple disease models including ALS, MS and PD (6), the utilization of iPS cell technology for drug abuse has been very limited. Our data suggests that as the proof of principle human iPSC-derived DA neurons may serve as a useful model to study human DA neurons in vitro and compare differences between control and drug dependent subjects. In the future, it might also be useful to test toxicity and pharmacological responses to potential treatments on an individual basis.