Ropinirole and Pramipexole Promote Structural Plasticity in Human iPSC-Derived Dopaminergic Neurons via BDNF and mTOR Signaling

Abstract

The antiparkinsonian ropinirole and pramipexole are D3 receptor- (D3R-) preferring dopaminergic (DA) agonists used as adjunctive therapeutics for the treatment resistant depression (TRD). While the exact antidepressant mechanism of action remains uncertain, a role for D3R in the restoration of impaired neuroplasticity occurring in TRD has been proposed. Since D3R agonists are highly expressed on DA neurons in humans, we studied the effect of ropinirole and pramipexole on structural plasticity using a translational model of human-inducible pluripotent stem cells (hiPSCs). Two hiPSC clones from healthy donors were differentiated into midbrain DA neurons. Ropinirole and pramipexole produced dose-dependent increases of dendritic arborization and soma size after 3 days of culture, effects antagonized by the selective D3R antagonists SB277011-A and S33084 and by the mTOR pathway kinase inhibitors LY294002 and rapamycin. All treatments were also effective in attenuating the D3R-dependent increase of p70S6-kinase phosphorylation. Immunoneutralisation of BDNF, inhibition of TrkB receptors, and blockade of MEK-ERK signaling likewise prevented ropinirole-induced structural plasticity, suggesting a critical interaction between BDNF and D3R signaling pathways. The highly similar profiles of data acquired with DA neurons derived from two hiPSC clones underpin their reliability for characterization of pharmacological agents acting via dopaminergic mechanisms.

Alpha6-Containing Nicotinic Acetylcholine Receptors Mediate Nicotine-Induced Structural Plasticity in Mouse and Human iPSC-Derived Dopaminergic Neurons

“…Human iPSCs from the F3 clone, previously characterized ( Collo et al, 2018 ), were induced to differentiate into floor plate (FP)-derived midbrain DA neurons using dual-SMAD inhibition and FP induction ( Kriks et al, 2011 ; Fedele et al, 2017 ) with minor modifications ( Collo et al, 2018 ). Human iPSCs were dissociated with Accutase TM (StemCell Technologies, Vancouver, BC, Canada), seeded (3 × 10 4 cells/cm 2 ) on Matrigel-coated plates in Knockout Serum Replacement (KSR) medium containing Knockout TM DMEM, 15% KSR, GlutaMAX TM , and 10 μM 2-mercaptoethanol, in the presence of LDN193189 (0.1 μM, Stemgent, Cambridge, MA, United States), SB431542 (10 μM, Tocris Bioscience), Shh C25II (0.1 μg/ml, R&D Systems), Purmorphamine (2 μM, Stemgent), Fibroblast Growth Factor 8 (0.1 μg/ml, R&D Systems), and CHIR99021 (3 μM, Stemgent).…”

“…The medium was changed to Neurobasal/B27/GlutaMAX TM supplemented with CHIR99021, BDNF (20 ng/ml, R&D Systems), ascorbic acid (AA; 0.2 mM, Sigma-Aldrich), dibutyryl cAMP (cAMP; 0.5 mM, Sigma-Aldrich), transforming growth factor type β3 (TGFβ3; 1 ng/ml, R&D Systems), glial cell line-derived neurotrophic factor (GDNF; 20 ng/ml, R&D Systems), and DAPT (10 nM, Tocris Bioscience). On day 21, cells were seeded on plates pre-coated with Polyornithine/Fibronectin/Laminin and co-cultured with mouse primary cortical astrocytes ( Collo et al, 2018 ) that were isolated and cultured according to Sorg and Magistretti (1991) . Human iPSC-derived DA neurons were used for pharmacological studies, immunofluorescence, immunocytochemistry, and morphological analysis starting from day 70.…”

“…A total of 3 days before pharmacological treatments, BDNF, AA, cAMP, TGFβ3, GDNF, and DAPT were gradually removed from the culture. Criteria to define DA neurons at day 70 were: neuronal morphology; co-expression of TH/MAP2; co-expression of TH/DAT; co-expression of TH/Vesicle monoamine associated transporter 2 (VMAT2); co-expression of TH/AMPAR subunit GluR2; and functional DA release and uptake from cultures containing DA neurons ( Collo et al, 2018 ). At this time, TH + /MAP2 + DA neurons were about the 30–40%, of the total MAP2 + neurons, in line with previous studies ( Deflorio et al, 2017 ; Fedele et al, 2017 ; Collo et al, 2018 ).…”

“…Criteria to define DA neurons at day 70 were: neuronal morphology; co-expression of TH/MAP2; co-expression of TH/DAT; co-expression of TH/Vesicle monoamine associated transporter 2 (VMAT2); co-expression of TH/AMPAR subunit GluR2; and functional DA release and uptake from cultures containing DA neurons ( Collo et al, 2018 ). At this time, TH + /MAP2 + DA neurons were about the 30–40%, of the total MAP2 + neurons, in line with previous studies ( Deflorio et al, 2017 ; Fedele et al, 2017 ; Collo et al, 2018 ). In addition, our cultures contained VGLUT2 + /MAP2 + neurons (25–30%) and GAD67 + /MAP2 + neurons (20–25%) ( Cavalleri et al, 2017 ; Collo et al, 2018 ).…”

“…However, relatively fewer studies were dedicated to investigate their response in pharmacological tests ( Cooper et al, 2012 ; Oni et al, 2016 ; Deflorio et al, 2017 ; Wang et al, 2018 ). We recently standardized a methodology to efficiently differentiate human iPSC into DA neurons and assess the effects of pharmacologic agents on structural neuroplasticity and intracellular signaling using morphological and biochemical techniques ( Fedele et al, 2017 ; Collo et al, 2018 ). In particular, in Collo et al (2018) , we studied the variability and reproducibility in iPSCs clones derived from healthy donors.…”