Integrating Target Prediction and Transcriptomic Analysis Identifies the AKR1C2-KRT8 Axis in Rotenone-Induced...

Parkinson's disease (PD) is a complex neurodegenerative disorder in which environmental toxins play a critical etiological role. Rotenone, a classical mitochondrial complex I inhibitor used to model PD, exerts its neurotoxicity through incompletely defined downstream molecular networks. Here, we integrated multiple PD transcriptomic datasets from GEO with predicted rotenone targets, applied machine learning to screen core candidate genes, and analyzed their cellular localization using single-cell transcriptomics. Molecular docking was performed to assess target-rotenone binding, and functional validation was carried out in primary dopaminergic neurons via lentivirus-mediated gene manipulation, Western blotting, qRT-PCR, and mitochondrial function assays. This approach identified and validated a six-gene core network (AKR1C2, AKR1C3, CES1, CTSS, DRD2, HSPA1A), several of which were predicted to directly bind rotenone. Single-cell analysis confirmed their enrichment in PD dopaminergic neurons, and immunofluorescence validated their co-localization with the dopaminergic marker TH. In rotenone-treated neurons, all six genes except DRD2, as well as KRT8, were significantly upregulated at both the mRNA and protein levels; rotenone also impaired mitochondrial Complex I activity and ATP production, and increased α-synuclein expression. In silico knockout revealed that AKR1C2-perturbed genes were enriched in keratinization pathways. Functional experiments demonstrated that AKR1C2 positively regulates KRT8. Notably, AKR1C2 knockdown not only reduced KRT8 levels but also rescued mitochondrial function, neuronal viability, and rotenone-impaired action potential firing, whereas AKR1C2 overexpression exacerbated these deficits. Critically, KRT8 re‑expression reversed the protective effects of AKR1C2 knockdown, while KRT8 knockdown reversed AKR1C2 overexpression‑induced impairments, collectively confirming the causal role of the AKR1C2-KRT8 axis. Collectively, these findings delineate a multi-node molecular network downstream of rotenone and provide the first experimental validation of a novel AKR1C2-KRT8 regulatory axis in dopaminergic neuron injury.