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Characterization of signaling pathways in in vitro developmental neurotoxicity battery (TSfN)
Citation:
Fitzpatrick, H., K. Wallace, T. Freudenrich, M. Mayil Vahanan, K. Carstens, AND T. Shafer. Characterization of signaling pathways in in vitro developmental neurotoxicity battery (TSfN). Presented at 9th Annual Triangle Society for Neuroscience (TSfN), Durham, NC, April 26, 2024.
Impact/Purpose:
Poster presented to 9th Annual Triangle Society for Neuroscience
Description:
Neurite outgrowth is an important precursor to synapse formation and assays for neurite outgrowth are part of an in vitro battery (IVB) of assays for developmental neurotoxicity screening (DNT). This battery of assays evaluates chemical effects on key neurodevelopmental processes to screen chemicals for potential DNT hazard. While positive outcomes in these assays can indicate a potential DNT hazard, interpretation and translation of these responses can be improved by understanding which important investigating the developmental signaling pathways that are expressed in the model systems comprising the DNT-IVB. Thus, to determine the presence of certain signaling pathways in two neurite outgrowth assays from the DNT-IVB, we tested various pathway modulators on neurite outgrowth (NOG) in human neurons (hNOG) and primary rat cortical tissue (rNOG). Human neurons (iCell GlutaNeurons; Fuji Cellular Dynamics) or rat primary cortex cultures, freshly prepared from post-natal day 0 rat cortex, were plated in triplicate on 96- well plates and dosed 2hrs later with a pathway modulator. Notch, Protein Kinase C (PKC), Nitric Oxide-cGMP, and Rho-associated protein kinase (ROCK) pathways were inhibited with DAPT, BIS-1, ODQ, and Y-27362, respectively, while WNT, PPAR-γ, and AKT/Protein Kinase B were activated with CHIR99021, pioglitazone, and SC79, respectively. All chemicals were tested from 1nM-30µM, except BIS-1 and CHIR99021 which were tested from 0.3nM-10µM and 0.8nM-25µM, respectively. Rac1 inhibitor (3, 10, and 30µM) and 0.1% DMSO were used as endpoint selective (positive) and negative controls. Forty-eight hours after dosing, samples were fixed and stained with Hoechst (nucleus) and Alexa Fluor 488-linked secondary antibodies against polyclonal rabbit β-tubulin (neurites) antibody. Plates were scanned using a Cellomics Array Scan, capturing the following endpoints: neuron count per valid field, neurite total length per neuron, and neurite total count per neuron. An active pathway was defined as producing 30% deviation in neurite total length and/or neurite count per neuron relative to DMSO control values. Neuron count per valid field was used as a metric for cell viability. In the hNOG and rNOG assays, the PKC inhibitor and WNT activator produced a marked decrease in neurite length and neurite count per neuron as a percent of control, without associated cytotoxicity. In both assays, the AKT activator also decreased the measured NOG endpoints but was accompanied by a precipitous decrease in viability. In both assays, PPAR-γ activator, ROCK inhibitor, NO-cGMP inhibitor, and Notch inhibitor were inactive and without cytotoxicity. The Notch inhibitor in the rNOG assay and the ROCK inhibitor in the hNOG assay were cytotoxic, decreasing the number of cells per field by 36% and 38% relative to control, respectively. These results demonstrate that PKC, WNT, and AKT pathways are active in both the human and rat models and play a role in neurite outgrowth. By contrast, Notch, NO-cGMP, PPAR-γ, and ROCK pathways did not appear to be necessary for neurite outgrowth in either model. However, inhibition of the Notch pathway in rat cells, and inhibition of the ROCK pathway in human neurons decreased cell viability, indicating that these pathways are important for basic functions impacting cell viability. Future studies using modulators of additional developmentally important pathways will expand our understanding of the critical pathways in the full set of EPA DNT-IVB assays, including synaptogenesis, network formation, proliferation, and apoptosis assays. This information is useful to understand the biological relevance of compounds that are either active or inactive in these NOG assays and help to identify potential gaps in the biology covered by assays in the DNT IVB. (This abstract does not reflect EPA policy.)