Event Abstract

Cell-type specific contribution to neuronal network (dys)function in neurodevelopmental disorders.

  • 1 Radboud University Nijmegen Medical Centre, Netherlands
  • 2 Donders Institute for Brain, Cognition and Behaviour, Radboud University, Netherlands
  • 3 Radboud University Nijmegen, Department of Cognitive Neurosciences, Netherlands

Motivation Neurodevelopmental disorders (NDDs), including autism spectrum disorders and intellectual disability are phenotypically and genotypically heterogeneous disorders. Great progress has been made over recent years towards the identification of NDD-related genes. A remaining challenge however, is to connect the genetic causes to processes that establish and/or modify neuronal circuit function. So far, NDDs have widely been associated with excitation and inhibition imbalances in neuronal networks. Additionally, astrocyte dysfunction has recently been implicated in the pathophysiology of NDDs. To understand how genes causative for NDDs affect the different cell-types that compose neuronal networks and, consequently, how cell types contribute to abnormal network organisation and communication, we opted for a human based cell culture model. To do so, we describe an approach to study the contribution of either glutamatergic or GABAergic neurons and astrocytes derived from WT and NDD patients to neuronal network function using Micro-Electrode Arrays (MEAs). Materials and methods The different cell-types used in this study were derived via controlled differentiation of Induced Pluripotent Stem Cells (hiPSC) into induced neurons (iNeurons). All hiPSCs were obtained by reprogramming fibroblasts of healthy controls or NDD patients using the Yamanaka factors1. To differentiate hiPSCs into iNeurons, hiPSCs were first transfected with a lentivirus containing a reverse tetracycline-controlled transcriptional activator (rtTA), and a second lentivirus containing a transgene with an rtTA regulated promoter. We used overexpression of Neurogenin 2 (Ngn2) to generate glutamatergic upper-layer cortical neurons2. Additionally, using overexpression of Achaete-Scute Family BHLH Transcription Factor 1 (Ascl1) and Distal-Less Homeobox 2 (Dlx2) we generated GABAergic neuronal subtypes of the telencephalon and diencephalon3. Glutamatergic neurons were either grown alone, or co-cultured with GABAergic neurons. As the maturation of these neurons is glia-dependent, neurons were cultured together with either rat or human astrocytes from 2 days in vitro (DIV) on. Rat astrocytes were isolated from E18 embryo’s as previously described4. HiPSC derived human astrocytes were differentiated using small molecules. We investigated the electrophysiological activity of NDD and WT neuronal networks using Micro-Electrode Arrays (MEA; Multi Channel Systems, Reutlingen, Germany). For high-throughput phenotyping, we used Multi Channel Systems 24-well MEA system and software. On a single-cell level, spontaneous inhibitory or excitatory postsynaptic currents were assessed in neuronal subtypes using whole-cell patch-clamp recordings. To investigate the contribution of GABAergic neurons to glutamatergic neuronal network activity on both the single-cell and network level, we blocked GABAergic signalling in the presence of Picrotoxin (PTX, 100 µM). MEA recordings were analysed using a custom software package named SpyCode5 developed in MATLAB© (The Mathworks, Natick, MA, USA). Whole-cell patch-clamp recordings were analysed using Clampfit 10.2 (Molecular Devices, CA, USA). Results To investigate whether a loss of function of NDD genes leads to impairments at the neural circuitry level in-vitro, we grow WT- or NDD patient-derived iNeurons on MEA. Already after two weeks in-vitro we find that neurons derived from healthy subjects displayed spontaneous electrophysiological activity mainly composed by random spikes and bursts. Late in development (i.e. from the fourth week on) the neuronal network showed high level of spontaneous activity as well as synchronous network bursts. From this time point on, neuronal network activity remained stable. Our data show that several genes implicated in NDDs play a role in normal glutamatergic neuronal network function and, as a consequence, showed a strong contribution to the abnormal network communication we found in human neuronal cultures carrying mutations in NDD genes. Neurons derived from these NDD patients established spiking activity during early network development as well as synchronous events involving most of the channels of the MEAs later in development. However, our results imply a difference between NDD-patients and controls in terms of connectivity and network organization. Figure 1 shows an example of altered network organisation between glutamatergic neurons derived from a healthy control and a patient with NDD. Interestingly, independent clustering analysis revealed that patient lines from the same NDD syndromes phenotypically clustered together, but clustered separately between NDD syndromes. In addition to the characterisation of NDD patient derived glutamatergic neurons, preliminary data show that also GABAergic neurons play an important role in establishing abnormal communication in human neuronal cultures carrying mutations in NDD genes by failing in fine-tuning of neuronal network synchronisation. Discussion This study was designed to investigate the cell-type specific functional contribution of either glutamatergic neurons, GABAergic neurons or astrocytes to neuronal network dysfunction in NDDs. To do so, we used three independent protocols to differentiate hiPSCs into either glutamatergic- or GABAergic iNeurons and astrocytes. The main advantage of generating these pure populations over protocols generating a mix, is the amount of control over the cell type specific composition of hiPSC cultures. Using this approach we can generate composite cultures with a specific ratio of glutamatergic versus GABAergic neurons, and decide which cell class carries the NDD mutation. Conclusion Through this translational approach we have been able to garner a greater understanding of cell-type specific contributions to pathophysiological network behaviour in specific NDDs. Furthermore our data on glutamatergic neurons indicate that neuronal network measurement of iNeurons on MEAs is a robust and sensitive method to perform genotype-phenotype analyses for NDDs and can be a powerful platform for drug screening assays.

Figure 1

References

1 Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676, doi:10.1016/j.cell.2006.07.024 (2006).
2 Zhang, Y. et al. Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron 78, 785-798, doi:10.1016/j.neuron.2013.05.029 (2013).
3 Yang, N. et al. Generation of pure GABAergic neurons by transcription factor programming. Nat Methods 14, 621-628, doi:10.1038/nmeth.4291 (2017).
4 Frega, M. et al. Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays. J Vis Exp, doi:10.3791/54900 (2017).
5 Bologna, L. L. et al. Investigating neuronal activity by SPYCODE multi-channel data analyzer. Neural networks : the official journal of the International Neural Network Society 23, 685-697, doi:10.1016/j.neunet.2010.05.002 (2010).

Keywords: Human Induced Pluripotent Stem Cells, neurodevelopmental disorder, activity impairments, iPS derived cell models, derived neurons

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: Mossink B, Frega M, Van Rhijn J, Linda K, Schoenmaker C, Keller J, Janssen S, Nadif Kasri N and Schubert D (2019). Cell-type specific contribution to neuronal network (dys)function in neurodevelopmental disorders.. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00013

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Received: 18 Mar 2018; Published Online: 17 Jan 2019.

* Correspondence:
Miss. Britt Mossink, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands, britt.mossink@radboudumc.nl
Dr. Dirk Schubert, Donders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, Netherlands, Dirk.Schubert@radboudumc.nl