(1005-B) A Novel Quantitative High Throughput Screening (qHTS) Assay for Human Neurotoxicity Using 3D-suspension Culture of Dopaminergic Neurons
Monday, February 5, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: The environment plays a major role in causing Parkinson’s disease (PD) and several other neurodegenerative diseases, however major chemical toxicants may await discovery. Screening chemical libraries to identify such neurotoxicants demands an experimental model coupled with an in vitro neurotoxicity assay capable of mimicking the in vivo actions of toxicants with sufficient sensitivity and high capacity. To meet this challenge, we developed a novel cell-based qHTS human neurotoxicity assay based on our prior findings. (1) LUHMES-derived dopaminergic neurons are highly sensitive to known neurotoxicants (Tong et al, J Appl Toxicol 37:167, 2017), many of which disrupted metal metabolism- and oxidative stress pathways (Tong et al. Chem Res Toxicol 31:127, 2018); (2) The MT1G gene is a sensitive biomarker in LUHMES neurons, responding dynamically to known neurotoxicants that disrupt these stress pathways (Tong et al, Neurotox Res 38:967, 2020); and (3) A 3D-suspension cell culture platform can enable qHTS assay to identify neurotoxicants in LUHMES neurospheroids (Tong et al, submitted, 2023). Specifically, we used the CRISP/R technology to engineer LUHMES cells by inserting a small HiBiT peptide tag into the N-terminus of the MT1G protein. The engineered cell line responds to neurotoxic chemicals by producing the HiBiT-tagged MT1G protein, detected as Nano-Glo™ Luciferase activity. We demonstrated that the engineered LUHMES dopaminergic neurons responded selectively to the chemicals to produce luminescent signal. For example, 150 nM ziram induced the engineered LUHMES neurons to produce a luciferase signal that is ~500-fold higher than the DMSO control. Using the engineered cell line, we developed a qHTS assay in a 3D-suspension culture platform to screen the LOPAC 1280 library using a Caliper robot. Among the 1280 compounds, we identified 64 chemicals that stimulated reporter activity, which also caused cytotoxicity. Interestingly, some chemicals induced reporter gene activity at much lower concentrations than they caused cytotoxicity, whereas others killed cells without inducing reporter activity. Therefore, this novel assay can identify chemicals that induce the metallothionein stress-response genes to cause the death of dopaminergic neurons, the underlying cause of neurodegenerative pathogenesis including PD. We plan to use this novel assay to screen large chemical libraries to identify environmental chemical causes of neurodegenerative diseases. (This research was supported in part by the Intramural research program of the NCATS and the Tox21 Program of NIEHS at NIH)