High-Content in vivo DNT Test Batteries using Cell-Specific Characterization of the Neurodevelopment and Synaptogenesis in C. elegans as NAMs

Neurodevelopmental disorders such as ADHD, autism, and learning disabilities are rising globally. In the US, 1 in 6 children suffer from some form of developmental disability. One potentially important factor in their increase is exposure to chemicals of unknown toxicity in the environment. While there are over 350,000 chemicals in commercial use and around 13,000 produced in large amounts, only a small percentage have known toxicological data. Current testing methods for developmental neurotoxicology (DNT) are mostly based on observing behavioral, physiological, and anatomical defects in small mammals. These studies are prohibitively time-consuming and expensive for testing the huge backlog of commercial chemicals, and may produce hard- to-interpret results due to the complexity of observed endpoints and differences between multiple laboratories. Additionally, many jurisdictions are limiting the numbers of animals that can be used for testing. New approach methodologies (NAMs) are a new paradigm in toxicology testing that bring together alternative approaches such as in silico predictions, cell culture, and microphysiological systems-based assays, plus alternative model animals such as zebrafish, drosophila, and C. elegans. While in silico and in vitro methods can provide high throughput testing at much lower costs than mammalian studies with easier to interpret and predictive results, in vitro methods still have substantial costs and complex culturing conditions and neither approach can recapitulate the effects of a substance on a complete developing organism. C. elegans is an ideal alternative animal model for DNT testing. It has a complete nervous system with a fully characterized connectome and neuronal lineages, most of the same important neurotransmitter systems as humans, is fast and easy to grow to maturity in large numbers, and is amenable to targeted genetic manipulation. We propose to use C. elegans in a comprehensive DNT test battery where we will track neuronal, synaptic, and behavioral changes as the organism develops from late larval to adult stage after early exposure to DNT toxicants. In Aim 1, we will develop novel reporter strains with fluorescent markers for GABAergic and dopaminergic neurons and synapses, expose them to reference chemicals, and monitor the behavior of growing animals in culture before imaging them on our high-content microfluidic imaging platform at L4 and D1 stages. Specific behavioral endpoints will be analyzed by ML-based approaches from the behavioral videos. The high-resolution whole-body 3D images from on-chip imaging will be analyzed by semi-automated methods for key DNT indicators such as neuronal loss and mislocalization, membrane blebbing, and defective or missing synapses. All these endpoints will be combined to find the dose of a chemical at which the point of departure from the control occurs. In Aim 2, we plan to investigate potential modes of action using expression analysis of developmental and stress genes. We will test a representative subset of chemicals from the DNT-DIVERS library to establish confidence in our model and lay the foundations for a fully automated low-cost, high-throughput DNT testing method to assess the risks of common chemicals.