Single-cell transcriptomic analysis reveals autophagy-associated transcriptional vulnerability of dopaminergic neurons after atrazine exposure.

Atrazine (ATZ) is a widely used triazine herbicide with potential neurotoxic effects, but the cell-type-specific responses of midbrain dopaminergic neurons to ATZ exposure remain poorly defined. In this study, we applied single-cell RNA sequencing to midbrain tissues from adult male mice exposed to ATZ for 28 days (a sub-acute exposure) to characterize transcriptional alterations associated with dopaminergic neuronal injury. Three mice per group were used for scRNA-seq, and group comparisons were performed using one-way ANOVA followed by Tukey's post hoc test, or the Kruskal-Wallis test with Dunn's post hoc test for non-normally distributed data. ATZ exposure reshaped the transcriptional profile of dopaminergic neurons and induced the activation of dopamine-related genes involved in dopamine synthesis, vesicular transport, reuptake, and receptor signaling, including Th, Slc18a2, Slc6a3, and Drd2. Programmed cell death module scoring revealed a significant increase in autophagy-associated signatures after ATZ exposure. Subpopulation analysis further showed that substantia nigra pars compacta dopaminergic neurons exhibited stronger programmed cell death and autophagy-related responses than ventral tegmental area dopaminergic neurons, indicating selective vulnerability within the midbrain dopaminergic system. Pseudotime trajectory analysis suggested a dynamic transition from dopamine-related gene activation to increased autophagy-related gene expression and altered neuronal state. Gene regulatory network inference computationally inferred a putative Th-Atg5 regulatory relationship linking dopaminergic functional disturbance with autophagy-associated signatures. Immunofluorescence validation confirmed increased Slc18a2 and LC3B expression in dopaminergic neurons, particularly in the substantia nigra pars compacta. These findings provide single-cell evidence that ATZ exposure is associated with a region-selective transcriptional vulnerability characterized by activation of dopamine-related genes, autophagy-associated vulnerability, and regional heterogeneity, offering new insight into the neurotoxic mechanisms of environmental herbicide exposure.