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

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.


Embryoid bodies generation and differentiation
Human iPSCs were mechanically dissociated and placed in suspension culture in hESC medium. After 8 days embryoid bodies (EBs) formed. At this stage EBs were plated into adherent conditions on Matrigel-coated dishes (for RNA extraction) or coverslides (for immunostaining) and maintained in culture 8 days to induce spontaneous differentiation.

Alkaline Phosphatase staining of hiPSCs
For the detection of Alkaline Phosphatase (AP), F3 human iPSCs were fixed with PBS containing 3% paraformaldehyde (2 min at RT) and incubated with Fast Red Violet Solution, Napthol AS-BI Phosphate Solution (all from Merck Millipore, Milan, Italy) and water in a 2:1:1 ratio. The samples were visualized using an Olympus IX51 microscope.

TaqMan Scorecard analysis of human iPSCs and embryoid bodies
Expression of pluripotency markers and determination of trilineage differentiation potential were evaluated for F3 hiPSCs and randomly differentiated cells from F3 EBs using the TaqMan hPSC Scorecard TM Panel kit (96 well, StepOne Plus; Thermo Fisher Scientific, MA) following the company's guidelines and using its cloud-based online analysis software. Total RNA was isolated using Quick-RNA MiniPrep (Zymo Research). Single-strand cDNA was obtained from 1 µg of total RNA by reverse transcription (RT) using the high-capacity cDNA RT Kit (Thermo Fisher Scientific).

Karyotype analysis
Cytogenetic studies were performed on chromosomes from CP01 human fibroblasts and from F3 hiPSCs after seven passages. Chromosome preparations were obtained according to standard techniques for cytogenetic samples adding 10 µg/ml Colcemid Solution (Irvine Scientific, Santa Ana, CA) in Hank's balanced salt solution (Sigma-Aldrich). 10-20 colonies were picked dissociated using Trypsin / EDTA (Sigma-Aldrich). Cells were incubated in hypotonic solution and then fixed in methanol and acetic acid (3:1). Metaphase suitable for analysis was sequentially Q-banded according to the routine methods.

RNA extraction and RT-PCR analysis
Total RNA from hiPSC-derived DA cultures was extracted using Quick-RNA MiniPrep (Zymo Research, Irvine, CA) according to the manufacturer's instructions.