Single-nucleus transcriptomics reveals chronic GenX exposure-induced neurotoxicity and predicted alterations in microglia-neuron communication networks in mouse brain

GenX (hexafluoropropylene oxide dimer acid, HFPO-DA), an emerging replacement for legacy per- and polyfluoroalkyl substances (PFAS), has raised increasing concerns regarding its potential health risks. However, the neurotoxic effects and underlying mechanisms of chronic GenX exposure remain poorly understood. In the present study, we established a chronic oral gavage mouse model of GenX exposure and combined targeted UPLC-MS/MS analysis, molecular biological validation, and single-nucleus RNA sequencing (snRNA-seq) to systematically investigate GenX exposure-induced significant motor and memory deficits in mice. Targeted UPLC-MS/MS analysis revealed accumulation of GenX in blood, liver, and brain tissues. In addition, GenX exposure disrupted both the intestinal barrier and blood–brain barrier, as evidenced by increased Evans blue extravasation and reduced expression of tight junction proteins. snRNA-seq analysis revealed broad transcriptional alterations across multiple brain cell populations. Neuronal populations exhibited enrichment of neurodegeneration- and apoptosis-related pathways, and abnormal expression of apoptosis-associated proteins. Moreover, microglia displayed inflammatory activation characterized by increased expression of activation-related genes, activation of NLRP3/Caspase-1 signaling, and altered pseudotime trajectories marked by decreased P2ry12 and increased Apoe expression. CellChat analysis further predicted alterations in microglia-neuron communication networks involving NRXN, NCAM, Cx3cl1-Cx3cr1, and Gas6-Mertk signaling pathways. Collectively, these findings demonstrate that chronic GenX exposure induces broad neurotoxic alterations involving barrier dysfunction, neuronal apoptosis, microglial inflammatory activation, and disrupted intercellular communication, providing new insights into the neurotoxic mechanisms of emerging PFAS substitutes.