Calcium activity-dependent generation and maintenance of mesodiencephalic dopaminergic neurons from murine and human pluripotent stem cells in the context of Parkinson’s Disease (DACaION)

• Applicants: Dr. Ralf Kühn; Professorin Dr. Nilima Prakash
• Subject Area: Molecular and Cellular Neurology and Neuropathology ; Developmental Neurobiology ; Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2018 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 408031320

The emergence of the cellular and molecular heterogeneity of dopamine-releasing (dopaminergic, DA) nerve cells in the midbrain during human and mouse embryonic development is largely not yet understood. These nerve cells participate in different cognitive, affective/emotional and motor brain functions. Their loss or dysfunction leads to severe neuropsychiatric human diseases, such as Parkinson’s Disease (PD), schizophrenias and addictive disorders. Our previous work has shown that a precise modulation of the canonical WNT/b-catenin signaling pathway is necessary and sufficient for the correct differentiation of a precursor population in the mouse midbrain into substantia nigra pars compacta (SNc) DA neurons. Moreover, the WNT/b-catenin signaling pathway promotes the survival of these neurons in a genetic mouse model of PD. The identification of the target genes of WNT/b-catenin signaling was therefore of particular interest in this context. The laser-microdissected WNT-responsive cells in the ventral midbrain of the mouse embryo express mostly genes encoding calcium (Ca2+) ion channel subunits, Ca2+ sensor proteins and other Ca2+-associated ion channels. Electrophysiological activities mediated by Ca2+ ions have already been demonstrated in the ventral midbrain of the mouse embryo at these early developmental stages.We now want to investigate the function of these Ca2+-mediated activities and Ca2+-associated ion channels and sensor proteins during the generation and maintenance of midbrain DA neurons in general and specific subsets of these neurons in particular. To this end, we want to monitor the Ca2+-mediated activities by Ca2+ imaging during the in vitro differentiation of human and mouse pluripotent stem cells into midbrain DA neurons, and to correlate these activities with the corresponding cell fate. For this purpose, we will use mouse embryonic (ES) and induced pluripotent (iPS) reporter stem cells as well as human iPS cells that have been equipped with subpopulation-specific reporter genes using CRISPR/Cas9 technology. In addition, we want to determine the function of particular Ca2+-associated ion channels and sensor proteins during this process by applying specific antagonists/blockers of these ion channels or by CRISPR/Cas9-mediated inactivation of the corresponding genes in the human iPS cells. We will also perform these analyses on differentiating iPS cells derived from idiopathic PD patients and healthy controls to obtain initial insights into potentially aberrant Ca2+ parameters in the PD-derived cells. Correction of such aberrant Ca2+ activities by the selective application of agonists/antagonists of these ion channels might be used for the development of new therapeutic approaches to PD.

• DFG Programme: Research Grants