Remote control of induced dopaminergic neurons in parkinsonian rats
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
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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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 2

Analysis of the reprogrammed neuronal phenotype of iDA neurons in vivo at different times of dox administration.

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Analysis of the reprogrammed neuronal phenotype of iDA neurons in vivo a...
(A) Schematic representation of lentiviral infection of iDA neurons before transplantation. Reprogrammed cells were FACS sorted on day 4 and replated at high density for tdTomato lentiviral infection on day 5. iDA neurons were left in culture for 24 hours and then grafted into the striatum of Rag2–/– IlrgtmWjl–null mice. (B–D) Immunohistochemical assay of grafted iDA neurons after 6 days of dox exposure in vitro. Immunostaining reveals the presence of tdTomato+TH-GFP– cells with fibroblast-like morphology. (E–G) tdTomato+ cells were also positively stained for collagen I, confirming their regression to fibroblasts. (H–J) Immunohistochemistry of grafted iDA neurons after 6 DIV plus 6 days in vivo of dox administration revealed the coexpression of both tdTomato and GFP reporters. A comparable result was obtained by extending dox administration in vivo from 6 to 12 days (K–M). Scale bars: 100 μm (B–D), 30 μm (E–G), 100 μm (H–M). B–M are representative images of 3 independent experiments performed on 3 mice per group.