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)
Developmental Neurobiology
Molecular Biology and Physiology of Neurons and Glial Cells
Final Report Abstract
Nerve cells located in the mammalian ventral midbrain (VM) and releasing the neuromodulator dopamine (DA), so-called midbrain dopaminergic (mDA) neurons, play pivotal roles in human brain function. Traditionally, the mDA neurons have been classified in two major subpopulations depending on their anatomical location in the VM and their functions. Thus, neurons located in the substantia nigra pars compacta (SNc) project primarily to the dorsolateral striatum and are involved in the control of voluntary motor behavior, whereas neurons located in the ventral tegmental area (VTA) innervate the prefrontal cortex and limbic areas of the brain, where they modulate cognitive and motivational/rewarding brain functions. Accordingly, degeneration or dysfunction of these mDA neurons lead to severe human neurodegenerative and neuropsychiatric disorders, such as Parkinson’s Disease (PD), schizophrenias and substance use disorders. However, it has meanwhile become clear that the mDA neurons exhibit a much broader cellular, molecular and physiological diversity than previously anticipated. So far, little if anything is known about how this mDA diversity is established during human pre- and early postnatal development, and how it impinges on the differential vulnerability of these neurons to, for example, degeneration in adult life. The main aim of this research project was to determine the expression and function of previously identified (in the mouse embryo) calcium (Ca2+)-related proteins and the corresponding Ca2+ activity-dependent processes in the directed differentiation of human induced pluripotent stem cells (hiPSCs) into mDA neurons and/or survival of these cells, and their diagnostic or therapeutic relevance in the context of PD. During the 3-year funding period, we generated, characterized and published a novel red fluorescent reporter hiPSC line (NES-mScarlet #18) for human neural stem cells (NSCs)/neural progenitor cells (NPCs) and other NESTIN-expressing cells. This new hiPSC line is a reliable reporter of NESTIN expression in NSCs/NPCs during mDA neuron differentiation in vitro, and can be used for the live cell imaging and fluorescence-based sorting of these cells. We also published our work about the transcriptomic analysis of the WNT/bcatenin-responding domain in the embryonic mouse VM, in which we showed that a dosedependent and subset-specific regulation of LEF1-mediated WNT/b-catenin signaling is necessary and sufficient for the proper differentiation of the mDA neurons in general (higher WNT/b-catenin doses inhibiting their differentiation into postmitotic mDA precursors and neurons), and of the SNc DA (arising from a domain with low or absent WNT/b-catenin signaling levels) and VTA DA (arising from a strong WNT/b-catenin signaling domain) neurons in particular. Our preliminary data indicate that a precise modulation of WNT/b-catenin signaling during human mDA NSC/NPC differentiation indeed allows the preferential in vitro generation of either SNc DA or VTA DA neurons. This is highly relevant for the modeling of PD and other mDA-related diseases in the culture dish and its therapeutic implications as well as for drug screening. Finally, we detected a statistically significant downregulation of three (3) out of 64 Ca2+ activity-related candidate genes in mature hiPSC-derived mDA neurons from idiopathic (sporadic) PD patients relative to their healthy controls. The corresponding genes will be followed up as pathophysiologically relevant for idiopathic PD in future research projects. This might be particularly interesting because a dysregulated Ca2+ homeostasis has been repeatedly demonstrated in hiPSC-derived mDA neurons from genetic (familial) PD cases at early stages of their differentiation.
Publications
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Crosstalk of Intercellular Signaling Pathways in the Generation of Midbrain Dopaminergic Neurons In Vivo and from Stem Cells. Journal of Developmental Biology, 7(1), 3.
Brodski, Claude; Blaess, Sandra; Partanen, Juha & Prakash, Nilima
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Dose-Dependent and Subset-Specific Regulation of Midbrain Dopaminergic Neuron Differentiation by LEF1-Mediated WNT1/b-Catenin Signaling. Frontiers in Cell and Developmental Biology, 8.
Nouri, Parivash; Götz, Sebastian; Rauser, Benedict; Irmler, Martin; Peng, Changgeng; Trümbach, Dietrich; Kempny, Christian; Lechermeier, Carina G.; Bryniok, Agnes; Dlugos, Andrea; Euchner, Ellen; Beckers, Johannes; Brodski, Claude; Klümper, Claudia; Wurst, Wolfgang & Prakash, Nilima
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Developmental pathways linked to the vulnerability of adult midbrain dopaminergic neurons to neurodegeneration. Frontiers in Molecular Neuroscience, 15.
Prakash, Nilima
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Generation of a NES-mScarlet Red Fluorescent Reporter Human iPSC Line for Live Cell Imaging and Flow Cytometric Analysis and Sorting Using CRISPR-Cas9-Mediated Gene Editing. Cells, 11(2), 268.
Nouri, Parivash; Zimmer, Anja; Brüggemann, Stefanie; Friedrich, Robin; Kühn, Ralf & Prakash, Nilima
