Human IPSC derived dopaminergic neurons show reduced activity following heterozygous FOXP2 knockout
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1
Radboud University Nijmegen Medical Centre, Department of Cognitive Neuroscience, Netherlands
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2
Donders Institute for Brain, Cognition and Behaviour, Radboud University, Netherlands
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3
Radboud University Nijmegen Medical Centre, Department of Human Genetics, Netherlands
Motivation
Heterozygous mutations of the transcription factor FOXP2 in humans lead to childhood apraxia of speech. FOXP2 is highly expressed in GABAergic and dopaminergic neurons throughout the basal ganglia, including expression in the striatum, substantia nigra and multiple thalamic nuclei. Neuronal dysfunction caused by impaired FOXP2 function could lead to a strong cellular phenotype within these areas. Individuals with heterozygous FOXP2 mutations indeed show impaired striatal grey matter volume and reduced striatal activity. Studies using mouse models for Foxp2 show that loss of Foxp2 function leads to impaired striatal development and aberrant striatal activity, specifically through a reduction of excitatory activity in striatal dopamine receptor type 1 expressing medium spiny neurons (MSNs). However, it is currently unknown if FOXP2 could cause similar functional deficits in human neurons. Human FOXP2 has been suggested to be functionally unique compared to FoxP2 from other species due to two human-specific amino-acid substitutions within functional domains [1]. We therefore investigated whether the cellular phenotypes present in mice with a heterozygous Foxp2 loss of function mutation could be present as well in human IPSC derived dopaminergic neurons.
Materials and methods
The dopaminergic neurons used in this study were derived via controlled differentiation of induced pluripotent stem cells (hiPSCs) into induced dopaminergic neurons (iNeurons). The hiPSCs were generated from reprogrammed fibroblasts of a healthy control using Yamanaka factors [2] and an isogenic line was produced in which a heterozygous knockout of FOXP2 was introduced. Dopaminergic neurons were generated from hiPSCs by addition of multiple combinations of small molecules and growth factors throughout the differentiation protocol (Figure 1A). Mature dopaminergic neurons were obtained after 55 days in vitro (DIV). We measured electrophysiological activity of wild-type and FOXP2+/- neuronal network using Multi-Electrode Arrays (MEA; Multi Channel Systems, Reutlingen, Germany) and by measuring spontaneous excitatory postsynaptic currents (sEPSCs) using whole cell patch clamp in mature dopaminergic neurons (DIV55 and 73). MEA recordings were analyzed using a custom software package (SpyCode [3]) developed in MATLAB (Mathworks, Natick, MA, USA). sEPSC activity was analyzed using Minianalysis 6.0.2 (Synaptosoft, USA). Statistical analysis was conducted in PRISM (Graphpad PRISM 7.00, Graphpad Software, San Diego, CA).
Results
We set out to conclusively validate the expression of Foxp2 in hiPSC derived dopaminergic iNeurons. Immunofluorescent staining of FOXP2 shows clear expression at DIV55. Thus, dopaminergic iNeurons can be used to assess FOXP2 function. iNeurons generated by this differentiation protocol display spontaneous excitatory activity already at DIV55, which strong synchronous network bursts at DIV73 (Figure 1B). When we compare the sEPSC activity and network activity at the MEA between wild-type and heterozygous knockout cells, a clear reduction in sEPSC amplitude and network bursts is visible at both DIV55 and DIV73. Intriguingly, we measured an increase in sEPSC frequency in cells from the FOXP2+/- line, which was highly prevalent at DIV73.
Discussion
Here we show that heterozygous knockout of FOXP2 reduces excitatory activity in neuronal cultures with a homogenous population of dopaminergic iNeurons. This is in line with the reduced excitatory activity which has been observed in dopamine receptor type 1 expressing striatal medium spiny neurons (MSNs) of mice with a heterozygous Foxp2 loss of function mutation. Intriguingly, we show an increase in sEPSC frequency in dopaminergic iNeurons, which is not described in heterozygous mice. This could be due to cellular identity, as striatal MSNs are GABAergic and do not form excitatory connections between each other. Striatal excitatory projections originate from the cortex, by cells which do not express FoxP2. Furthermore, changes in inhibitory activity are profoundly present in striatal MSNs of heterozygous Foxp2 loss of function mice. Therefore, the generation of inhibitory neurons which express FOXP2 will be an essential next step to further explore the cellular phenotypes related to FOXP2 in a human context.
Conclusion
The generation of specific cell types which have been implicated in neurodevelopmental disorders is a viable translational tool to extend previous findings discovered using mouse models to the human situation. The overlap of cellular phenotypes between mouse brain and human IPSC derived neurons with Foxp2 loss of function mutations suggests molecular and physiological mechanisms are similarly affected, reinforcing iNeurons as a viable platform for6 disease modeling.
References
References
References
[1] G. Konopka, J.M. Bomar, K. Winden, G. Coppola, Z.O. Jonsson, F. Gao, S. Peng, T.M. Preuss, J.A. Wohlschlegel, and D.H. Geschwind, Human-specific transcriptional regulation of CNS development genes by FOXP2. Nature 462 (2009) 213-7.
[2] K. Takahashi, and S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126 (2006) 663-76.
[3] L.L. Bologna, V. Pasquale, M. Garofalo, M. Gandolfo, P.L. Baljon, A. Maccione, S. Martinoia, and M. Chiappalone, Investigating neuronal activity by SPYCODE multi-channel data analyzer. Neural Netw 23 (2010) 685-97.
Keywords:
dopaminergic neuron,
FoxP2,
human induced pluripotent stem cell,
Disorder modeling,
activity impairments
Conference:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.
Presentation Type:
Poster Presentation
Topic:
Stem cell-derived applications
Citation:
Van Rhijn
J,
Shi
Y,
Nadif Kasri
N and
Schubert
D
(2019). Human IPSC derived dopaminergic neurons show reduced activity following heterozygous FOXP2 knockout.
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00118
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Received:
18 Mar 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
Mr. Jon-Ruben Van Rhijn, Radboud University Nijmegen Medical Centre, Department of Cognitive Neuroscience, Nijmegen, Netherlands, jon-ruben.vanrhijn@radboudumc.nl
Dr. Dirk Schubert, Radboud University Nijmegen Medical Centre, Department of Cognitive Neuroscience, Nijmegen, Netherlands, Dirk.schubert@radboudumc.nl