Neurodevelopment functional alterations caused by per- and poly-fluoroalkyl substances (PFAS) investigated in in vitro human dopaminergic models

Per- and polyfluoroalkyl substances (PFAS) are synthetic compounds known as “forever chemicals” for their tendency to bioaccumulate. Their extensive industrial and commercial use has led to widespread contamination of water and soil, with PFAS detectable in different tissues of the human body. We previously reported a significant accumulation of perfluorooctanoic acid (PFOA) in the substantia nigra of individuals from highly contaminated areas in the Veneto region [Di Nisio et al., 2022]. Since this brain region is rich in dopaminergic neurons (DANs) that are particularly sensitive to aging and environmental toxicants, PFAS bioaccumulation may contribute to dopaminergic dysfunction. This study aims to investigate how environmentally relevant legacy PFAS affect the development and function of human DANs. To this end, we combined 2D LUHMES and iPSC-derived DAN cultures with 3D human midbrain organoids and exposed them to PFOA across a concentration range spanning environmental to occupational levels (1–10000 ng/ml). We integrated advanced biophysical approaches (patch clamp, MEA recordings and two-photon calcium imaging) with molecular and biochemical assays to investigate the impact of PFOA exposure on human dopaminergic models. We found that, despite preserved cell viability, PFOA reduced Na+ currents, enhanced K+ currents, broadened and attenuated action potentials, consistent with impaired intrinsic excitability. At the network level, these changes were associated with a dose-dependent reduction in mean firing rate, loss of synchrony, and elevated intracellular Ca2+. Molecular analyses revealed a reduction of TH expression and dopamine release and downregulation of synaptic markers, consistent with impaired dopaminergic transmission. Overall, our findings indicate that PFOA at concentrations comparable to those measured in exposed populations does not kill DANs but instead dampens their functional output. This supports a scenario in which “forever chemicals” increase the risk of dopamine-related neurodevelopmental and neurodegenerative disorders by gradually reshaping dopaminergic circuits rather than triggering acute degeneration.