Image-Based Quantitation of Kainic Acid-Induced Excitotoxicity as a Model of Neurodegeneration in Human iPSC-Derived Neurons

Talbot, Jana; Chear, Sueanne; Phipps, Andrew J.; Pébay, Alice; Hewitt, Alex W.; Vickers, James C.; King, Anna E.; Cook, Anthony

doi:10.1007/7651_2021_421

Cited by 5 publications

(2 citation statements)

References 30 publications

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“…4-AP is a K + channel blocker that stimulates the release of both excitatory and inhibitory neurotransmitters in neurons both in vitro and in vivo and is known to increase glutamatergic transmission and cause seizures or convulsions [ 27 ]. KA is an excitatory toxic substance that stimulates the glutamate receptor and causes neuronal cytotoxicity and is often used in seizure models in vitro and in vivo [ 28 , 29 , 30 ]. Acetaminophen is a medication used to treat fever and pain with little incidence of seizure in humans with therapeutic doses.…”

Section: Methodsmentioning

confidence: 99%

High-Throughput Screening Assay for Detecting Drug-Induced Changes in Synchronized Neuronal Oscillations and Potential Seizure Risk Based on Ca2+ Fluorescence Measurements in Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neuronal 2D and 3D Cultures

Seo

Kreir

et al. 2023

Cells

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Drug-induced seizure liability is a significant safety issue and the basis for attrition in drug development. Occurrence in late development results in increased costs, human risk, and delayed market availability of novel therapeutics. Therefore, there is an urgent need for biologically relevant, in vitro high-throughput screening assays (HTS) to predict potential risks for drug-induced seizure early in drug discovery. We investigated drug-induced changes in neural Ca2+ oscillations, using fluorescent dyes as a potential indicator of seizure risk, in hiPSC-derived neurons co-cultured with human primary astrocytes in both 2D and 3D forms. The dynamics of synchronized neuronal calcium oscillations were measured with an FDSS kinetics reader. Drug responses in synchronized Ca2+ oscillations were recorded in both 2D and 3D hiPSC-derived neuron/primary astrocyte co-cultures using positive controls (4-aminopyridine and kainic acid) and negative control (acetaminophen). Subsequently, blinded tests were carried out for 25 drugs with known clinical seizure incidence. Positive predictive value (accuracy) based on significant changes in the peak number of Ca2+ oscillations among 25 reference drugs was 91% in 2D vs. 45% in 3D hiPSC-neuron/primary astrocyte co-cultures. These data suggest that drugs that alter neuronal activity and may have potential risk for seizures can be identified with high accuracy using an HTS approach using the measurements of Ca2+ oscillations in hiPSC-derived neurons co-cultured with primary astrocytes in 2D.

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Section: Methodsmentioning

confidence: 99%

High-Throughput Screening Assay for Detecting Drug-Induced Changes in Synchronized Neuronal Oscillations and Potential Seizure Risk Based on Ca2+ Fluorescence Measurements in Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neuronal 2D and 3D Cultures

Seo

Kreir

et al. 2023

Cells

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“…Neural induction and differentiation were done essentially as described [65]. When the iPSCs were 80% confluent, cells were dissociated into small clumps using Accutase (Gibco) and seeded at a density of 3 × 10 5 cells/well in poly-l-ornithine (PLO, Sigma)/laminin (Gibco)-coated 6-well plate.…”

Section: Methodsmentioning

confidence: 99%

Lysosomal alterations and decreased electrophysiological activity in CLN3 disease (966 bp deletion, E295K) patient-derived cortical neurons

Chear

Perry

Wilson

et al. 2022

Preprint

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CLN3 disease is a lysosomal storage disorder associated with fatal neurodegeneration that is caused by mutations in CLN3. Most individuals with CLN3 disease carry at least one allele with a 966 bp deletion in CLN3 which results in the deletion of exons 7 and 8. There is a need for more physiologically relevant human cell-based CLN3 disease models to better understand the cellular changes during the disease process. Using CRISPR/Cas9, we corrected the 966 bp deletion mutation in human induced pluripotent stem cells (iPSCs) of a compound heterozygous patient (CLN3 Δ 966 bp and E295K). The isogenic deletion-corrected and unedited CLN3 patient iPSCs were used for disease modeling. iPSC-derived neurons carrying this particular CLN3 mutation (CLN3 neurons) had lower functional activity as recorded using microelectrode arrays for most of the culture period. Proteomics analysis showed downregulation of proteins related to axon guidance and endocytosis at day in vitro (DIV) 14 and 42 in CLN3 neurons. This was accompanied by an increase in lysosomal-related proteins in CLN3 neurons. Western blot analysis revealed hyperglycosylation of the lysosomal marker, Lysosome Associated Membrane Protein 1 (LAMP1) in CLN3 neurons at DIV 14, 28 and 42, which was not apparent in control neurons. Ultrastructural analysis of CLN3 neurons showed numerous membrane-bound vacuoles containing diverse types of storage material, ranging from curvilinear deposits, multilamellar structures to osmiophilic deposits. Our findings suggest alterations in lysosomal function and neurodevelopment involving axon guidance and synaptic transmission in CLN3-deficient neuronal derivatives, which could be potential targets for therapy.

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How is Excitotoxicity Being Modelled in iPSC-Derived Neurons?

Cheng,

Cook,

Talbot

et al. 2024

Neurotox Res

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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.

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Image-Based Quantitation of Kainic Acid-Induced Excitotoxicity as a Model of Neurodegeneration in Human iPSC-Derived Neurons

Cited by 5 publications

References 30 publications

High-Throughput Screening Assay for Detecting Drug-Induced Changes in Synchronized Neuronal Oscillations and Potential Seizure Risk Based on Ca2+ Fluorescence Measurements in Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neuronal 2D and 3D Cultures

High-Throughput Screening Assay for Detecting Drug-Induced Changes in Synchronized Neuronal Oscillations and Potential Seizure Risk Based on Ca2+ Fluorescence Measurements in Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neuronal 2D and 3D Cultures

Lysosomal alterations and decreased electrophysiological activity in CLN3 disease (966 bp deletion, E295K) patient-derived cortical neurons

How is Excitotoxicity Being Modelled in iPSC-Derived Neurons?

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