Integrating transcriptomics and molecular AOPs to identify T3-dependent regulation of neuronal development in the human neural progenitor test

Thyroid hormone (TH) is an important regulator of human brain development, and maternal TH imbalance is linked to adverse outcomes such as reduced IQ and decreased motor function in children. As part of the VHP4Safety initiative, using the human neural progenitor test (hNPT), we investigated the molecular effects of elevated levels and absence of T3. Neural progenitor cells were differentiated for 10 days under no (0 nM), normal (4.1 nM; the reference), or high (410 nM) triiodothyronine (T3) conditions. RNA-Seq was used to assess gene expression, and a molecular Adverse Outcome Pathway (AOP) network was applied to interpret transcriptomic changes compared to the reference alongside Gene Ontology (GO) enrichment analysis. Data show that normal T3 levels induce key neurodevelopmental processes which are not active without T3 exposure, based on significantly altered gene expression in pathways related to neurogenesis, synaptogenesis, differentiation and metabolism, including pathways of brain-derived neurotrophic factor (BDNF) signalling, proliferation, oligodendrocyte specification, and glycolysis. Key Events (KEs) such as reduced BDNF and impaired proliferation were, compared to normal levels of T3, significantly affected in the molecular AOP network in absence of T3, while high T3 levels showed minimal differences in transcriptional effects. Our findings demonstrate that absence of T3 inhibits brain development since no T3 condition affects key neurodevelopmental processes in vitro. This study illustrates the utility of integrating transcriptomics with molecular AOPs to provide a structured, mechanistic framework for evaluating the regulation of neuronal cell proliferation and differentiation in brain development. The framework may be useful for chemical risk assessment.