Remote control of induced dopaminergic neurons in parkinsonian rats
Maria Teresa Dell’Anno, … , Alexander Dityatev, Vania Broccoli
Maria Teresa Dell’Anno, … , Alexander Dityatev, Vania Broccoli
Published June 17, 2014
Citation Information: J Clin Invest. 2014;124(7):3215-3229. https://doi.org/10.1172/JCI74664.
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Research Article

Remote control of induced dopaminergic neurons in parkinsonian rats

Abstract

Direct lineage reprogramming through genetic-based strategies enables the conversion of differentiated somatic cells into functional neurons and distinct neuronal subtypes. Induced dopaminergic (iDA) neurons can be generated by direct conversion of skin fibroblasts; however, their in vivo phenotypic and functional properties remain incompletely understood, leaving their impact on Parkinson’s disease (PD) cell therapy and modeling uncertain. Here, we determined that iDA neurons retain a transgene-independent stable phenotype in culture and in animal models. Furthermore, transplanted iDA neurons functionally integrated into host neuronal tissue, exhibiting electrically excitable membranes, synaptic currents, dopamine release, and substantial reduction of motor symptoms in a PD animal model. Neuronal cell replacement approaches will benefit from a system that allows the activity of transplanted neurons to be controlled remotely and enables modulation depending on the physiological needs of the recipient; therefore, we adapted a DREADD (designer receptor exclusively activated by designer drug) technology for remote and real-time control of grafted iDA neuronal activity in living animals. Remote DREADD-dependent iDA neuron activation markedly enhanced the beneficial effects in transplanted PD animals. These data suggest that iDA neurons have therapeutic potential as a cell replacement approach for PD and highlight the applicability of pharmacogenetics for enhancing cellular signaling in reprogrammed cell–based approaches.

Authors

Maria Teresa Dell’Anno, Massimiliano Caiazzo, Damiana Leo, Elena Dvoretskova, Lucian Medrihan, Gaia Colasante, Serena Giannelli, Ilda Theka, Giovanni Russo, Liudmila Mus, Gianni Pezzoli, Raul R. Gainetdinov, Fabio Benfenati, Stefano Taverna, Alexander Dityatev, Vania Broccoli

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Figure 1

Morphological and functional stability of the reprogrammed state of iDA neurons in vitro.

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Morphological and functional stability of the reprogrammed state of iDA ...
(A) Scheme showing the experimental procedures for coculturing iDA cells and primary hippocampal neurons in the absence of dox. iDA neurons were FACS sorted on day 4 and directly plated onto primary cell cultures. From day 8, dox was removed, and cells were left in culture until day 21, when morphological and functional analyses were performed. LV, lentivirus. (B–D) Immunocytochemical analysis of iDA neurons in vitro after dox withdrawal. iDA neurons exhibited a differentiated morphology and long projections, with SYT1 and HOMER1+ puncta located along neurites. (E and F) Immunodetection of VMAT2 and its functional assessment on iDA neurons through FFN511. (G) Current-induced and spontaneous trains of APs recorded in iDA neurons at 21 DIV. (H) Spontaneous glutamatergic EPSCs at 21 DIV in iDA neurons recorded in voltage-clamp mode (Vh: –60 mV; top trace). After application of the AMPA receptor antagonist NBQX (5 μM), the EPSCs were completely blocked (bottom trace). Inset shows an averaged EPSC in an expanded time scale (n = 20 spontaneous events). Histograms show the distributions of the basic EPSC parameters. Scale bars: 50 μm (B), 10 μm (C–F). Data shown in B–H are representative of 3 independent experiments performed on 6 samples.