How is Excitotoxicity Being Modelled in iPSC-Derived Neurons?

Abstract

Excitotoxicity linked either to environmental causes (pesticide and cyanotoxin exposure), excitatory neurotransmitter imbalance, or to intrinsic neuronal hyperexcitability, is a pathological mechanism central to neurodegeneration in amyotrophic lateral sclerosis (ALS). Investigation of excitotoxic mechanisms using in vitro and in vivo animal models has been central to understanding ALS mechanisms of disease. In particular, advances in induced pluripotent stem cell (iPSC) technologies now provide human cell-based models that are readily amenable to environmental and network-based excitotoxic manipulations. The cell-type specific differentiation of iPSC, combined with approaches to modelling excitotoxicity that include editing of disease-associated gene variants, chemogenetics, and environmental risk-associated exposures make iPSC primed to examine gene-environment interactions and disease-associated excitotoxic mechanisms. Critical to this is knowledge of which neurotransmitter receptor subunits are expressed by iPSC-derived neuronal cultures being studied, how their activity responds to antagonists and agonists of these receptors, and how to interpret data derived from multi-parameter electrophysiological recordings. This review explores how iPSC-based studies have contributed to our understanding of ALS-linked excitotoxicity and highlights novel approaches to inducing excitotoxicity in iPSC-derived neurons to further our understanding of its pathological pathways.

Keywords: AMPAR; Calcium; DREADDs; Glutamate; Kainic acid; NMDAR.

Fig. 1

Generating excitotoxicity in ALS disease models. Excitotoxicity can be induced through application of glutamate, NMDA, and other structurally similar glutamate receptor agonists, such as KA and beta-methylamino-L-alanine (L-BMAA), that bind with NMDA and AMPA/KA receptors. Mutations to ALS-associated genes such as SOD1, C9ORF72, TARDBP, and FUS, lead to upregulated expression of glutamate receptors, increasing neuron sensitivity to glutamate-mediated activation. Excitotoxicity can also be induced in neurons through non-glutamate receptor pathways, such as through chloropyrifos-induced, indirect activation of cholinergic receptors

Fig. 2

Schematic depicting subunit formations of NMDA, AMPA, KA, and GABA receptors. NMDA and AMPA receptor subunits dictate cellular properties including calcium permeability and receptor conductance, where subunit expression levels can alter neuron susceptibility to excitotoxicity (Cull-Candy and Leszkiewicz ; Herguedas et al. 2016). While KA receptors are understood to form homomers and heteromers, the properties of each receptor subunit are not fully known (Hansen et al. ; Watanabe-Iida et al. 2016)

Fig. 3

Gene expression levels of neurotransmitter receptor subunits in iPSC-derived cortical neurons. Human iPSC cell line TOB-00220 was maintained in mTeSR Plus medium, and differentatied to PAX6- and Nestin-positive neural stem cells (NSCs), that were then differentiated to MAP2- and TAU-postive neurons as we have described previously (Chear et al. ; Talbot et al. 2021). RNA was extracted and RT-qPCR performed using TaqMan probes and analysed according to Chear et al. (2022) to demonstrate levels of neurotransmitter receptor subunit mRNA expression in iPSCs, NSCs, and neurons at 2, 4, and 6 weeks maturation. NMDA, AMPA, KA, and GABA receptor subunits were upregulated in neurons compared to iPSCs and NSCs. n = 3 biological replicates, data presented as mean ± standard error of the mean. The TOB-00220 iPSC line used for these experiments was generated previously (Daniszewski et al. 2022), and verified using a 10-panel short-tandem repeat analysis in March 2022 using services from the Australian Genome Research Facility

Fig. 4

iPSC-derived cortical neurons express functional neurotransmitter receptors that can be modulated by known neurotransmitter antagonists. Human iPSC cell line TOB-00198 was maintained in mTeSR Plus medium, and differentatied to PAX6- and Nestin-positive neural stem cells (NSCs), that were then differentiated to MAP2- and TAU-postive neurons as we have described previously (Chear et al. ; Talbot et al. 2021). MEA recordings were obtained and analysed essentially as we have described previously (Chear et al. 2022), using 12-electrode 24-well plates from Multichannel Systems. Neuronal activity was recorded for 10 min at 37 °C, following a 2 min equilibration, and data processed using threshold parameters for determination of spikes, and bursts. Antagonists were applied during recording, by adding equal volume of medium with 2 × final concentration. A) iPSC-derived neurons exhibit spontaneous action potential firing, which was altered by treatment with NMDA (dAP5, 10 µM), AMPA (CNQX, 25 µM; NBQX, 10 µM), and GABA (bicuculline, 10 µM ; picrotoxin, 100 µM) antagonists. B, C, D) Application of dAP5, CNQX, and NBQX attenuated neuronal activity, through a reduced spike rate, burst count, and burst duration, while treatment with bicuculline and picrotoxin increased neuronal activity. E, F) Treatment of iPSC-derived neurons with bicuculline and picrotoxin increased burst spike count and decreased interburst interval. Circles represent values of individual electrodes; error bars represent mean and standard error. The TOB-00198 iPSC line used for these experiments was generated previously (Daniszewski et al. 2022), and verified using a 10-panel short-tandem repeat analysis in March 2022 using services from the Australian Genome Research Facility

Fig. 5

Modulation of neuronal activity using DREADDs. DREADDs can be expressed in neurons to increase or decrease neuronal firing, depending on the subclass of DREADD expressed. DREADDs are designed to bind specifically with ligands not typically found in the natural environment of neurons, such as CNO and SALB. DREADDs, while derived from human muscarinic receptors, cannot interact with endogenously found neurotransmitters such as glutamate or other ligands, allowing for specific control of neuronal activity